Advances in Anatomic Pathology:
Diffuse Aggressive B-cell Lymphomas
Said, Jonathan MD
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine UCLA, Los Angeles, CA
Reprints: Jonathan Said, MD, UCLA CHS 13-226, 10833 Le Conte Avenue, Los Angeles, CA 90095 (e-mail: email@example.com).
Diffuse aggressive B-cell lymphomas comprise a relatively common and increasingly diverse group of neoplasms. Newer modalities including gene expression profiling and an increasing panel of immunohistochemical markers have contributed to greater accuracy in defining these entities. Attention is paid not only to the neoplastic cells but also to the cellular and stromal milieu in which they proliferate. These distinctions may have therapeutic implications as well, with improved outcome related to newer and sometimes targeted therapies. At the same time there is increasing understanding of the overlap, which occurs in the grey zone between diffuse large B-cell lymphoma and Burkitt lymphoma as well as between diffuse large B-cell lymphoma and Hodgkin lymphoma. This review aims to provide practical insights in the correct identification and differential diagnosis of these lymphomas, with emphasis on the changes that have occurred with the publication of the 2008 World Health Organization updated classification.
Diffuse aggressive B-cell lymphomas comprise a spectrum of lymphoproliferative disorders, which have been historically characterized according to the size of the proliferating malignant cells. Diffuse large B-cell lymphoma (DLBCL) has traditionally been defined as a lymphoid neoplasm in which the nucleus is equal to or exceeds the size of a normal macrophage nucleus or more than twice the size of a small lymphocyte. In contrast in cases of Burkitt lymphoma (BL) the nucleus is slightly smaller than a histiocyte nucleus. Despite their heterogeneity, diffuse aggressive B-cell lymphomas have features in common including the diffuse proliferation of intermediate-to-large B-lymphoid cells and a rapid proliferation rate. If untreated they are usually fatal within weeks to a few years of presentation, but they are potentially curable with current therapy modalities. Table 1 is a summary of the key clinical distinctions between BL and DLBCL.
DIFFUSE LARGE B-CELL LYMPHOMA
DLBCL incorporates an increasingly complicated spectrum of lymphoid neoplasms with markedly different histologic appearance, phenotype, and clinical behavior. The addition of rituximab to combination chemotherapy has significantly improved outcome.1–3 To correctly diagnose these entities a knowledge of the morphologic, genetic, and immunophenotypic diversity as well as differential diagnosis is essential.
DLBCL is the most common histologic subtype of non-Hodgkin lymphoma, and comprises greater than 30% of adult non-Hodgkin lymphoma in the West, and an even higher percentage in developing countries. DLBCL occurs at any age including childhood, with the median presentation in the seventh decade at an of age 64. It is slightly more common in males. About 40% of DLBCL are extranodal stage I or II, or patients may present with a nodal rapidly enlarging mass. The most common sites of involvement are the stomach, ileocecal region, bone, testis, spleen, Waldeyer's ring, salivary gland, thyroid, liver, kidney, and adrenal. The long-term remission rate is from 50% to 60% and has been improved with rituximab therapy, first used in elderly patients with DLBCL.1–3 The overall 5-year survival is 46%, and the failure-free survival is 41%. Unfavorable factors in DLBCL include older age >60 years, poor performance status (Eastern Cooperative Oncology Group >2), advanced stage (III-IV), extranodal involvement >2 sites, and high serum lactate dehydrogenase. In general, DLBCL share the characteristic of being spontaneously aggressive, and even with improved therapy in recent decades, about 40% remain uncured. Immunodeficiency is a risk factor for DLBCL, and about 10% of DLBCL are Epstein-Barr virus (EBV)+.4–6 The incidence of EBV association increases with increasing age.
Pathogenesis of Diffuse Large B-cell Lymphoma
DLBCL can arise de novo, or as a progression or transformation event in a patient with low grade lymphoma, including:
1. Chronic lymphocytic leukemia/small lymphocytic lymphoma (Richter's syndrome)
2. Follicular lymphoma
3. Marginal zone lymphoma
DLBCL is therefore a final common pathway in many patients who are long-term survivors of indolent lymphoma. In particular T-cell rich B-cell lymphoma (TCRBCL) has been associated with nodular lymphocyte predominant Hodgkin lymphoma.
Transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma to DLBCL is commonly called Richter's syndrome. Of clinical importance is classification of diffuse large cell lymphoma in patients with grade 3 follicular lymphoma. If there are diffuse areas containing greater than 15 centroblasts per high power field, then a separate diagnosis of DLBCL should be rendered, and this should precede the diagnosis of follicular lymphoma. For example in a lymph node that shows features of grade 3A or 3B follicular lymphoma and diffuse proliferation the diagnosis should read as:
DLBCL in X% of the lymph node, and follicular lymphoma grade 3 in Y% of the node. Staining for follicular dendritic cells with antibodies to CD21 or CD23 may be helpful in highlighting areas of follicular proliferation in a partially diffuse infiltrate (Fig. 1A).
Histologic Features of Diffuse Large B-cell Lymphoma
DLBCL is characterized by diffuse nodal architectural effacement by sheets of large cells of B-cell phenotype. Small T-lymphocytes/histiocytes are usually present, unlike BLs, which are more homogeneous and uniform. In some distinct subtypes such as TCRBCL, the background small lymphocytes and histiocytes may outnumber the large B-cells. TCRBCL is a distinct entity described below, and this diagnosis should not be made only because there are large numbers of reactive T-cells. Sclerosis is variable but may be prominent and mitotic figures are easily identified.
The World Health Organization (WHO) 2008 classification of DLBCL has been expanded to include a number of clearly defined as well as still putative entities with specific histologic, phenotypic, cytogenetic, and molecular genetic features7 (Table 2).
Difficulties With Diagnosis of Diffuse Large B-cell Lymphoma and Differential Diagnosis
Although the diagnosis of DLBCL is not difficult when there are sheets of malignant B-cells, often rapidly proliferating with numerous mitotic figures, there are situations in which the diagnosis can be problematic. In particular DLBCL may form cohesive sheets or involve sinusoids, mimicking carcinoma or melanoma. Nasopharyngeal carcinomas, neuroendocrine carcinomas, germ cell tumors (seminomas), and granulocytic sarcomas (chloromas) can all be mistaken for DLBCL and should be considered in the differential diagnosis. These can be usually identified using a standard panel of immunohistochemical markers including a keratin antibody such as AE1/3 or CAM5.2, neuroendocrine markers such as neuron-specific enolase, synaptophysin, chromogranin, germ cell tumor markers such as PLAP and OCT3/4, and melanoma markers such as HMB45 and Mart1.
In some cases there may be aberrant phenotypes that make DLBCL difficult to characterize (for example plasmablastic DLBCL with late differentiation often lacks CD20, as may anaplastic lymphoma kinase (ALK) positive DLBCL or DLBCL from patients who have been treated with rituximab). DLBCL may also share histologic or phenotypic features with other lymphomas, probably owing to common pathogenic pathways (for example Burkitt-like, Hodgkin-like, and grey zone lymphomas), which are discussed below. Some DLBCL may contain Hodgkin and Reed Sternberg (RS) cells, particularly those that are associated with immunodeficiency and infection with EBV. Blastoid mantle cell lymphomas can also be confused with DLBCL.
THE ROLE OF IMMUNOHISTOCHEMISTRY IN DIFFUSE LARGE B-CELL LYMPHOMA
DLBCL characteristically express pan B-cell markers including CD19, D20, CD22, CD79a, and PAX5 (Table 3). Surface/cytoplasmic immunoglobulin (IgM>IgG>IgA) is present in 50% to 75% of cases. An unusual circumstance is seen in ALK positive DLBCL that uniformly express clonal cytoplasmic IgA. CD30 expression is variable,8 but may be diffusely positive requiring differentiation from ALK negative anaplastic large cell lymphomas which are T/natural killer (T/NK) cell neoplasms. Unlike Hodgkin lymphoma DLBCL express a complete B-cell program including expression of transcription factors such as OCT-2 and BOB.19 Expression of LM02 is associated with the germinal center phenotype and associated with a more favorable prognosis, whereas expression of FOXP1 is associated with the activated B-cell (ABC) phenotype and more aggressive disease.10 Expression of T-cell markers are characteristically negative, although CD5+ and CD3+ variants of DLBCL have been described. Other markers variably expressed in DLBCL include CD10 (40%), BCL6 (60%), BCL2 (50%), CD43 (20%), CD5 (<10%), CD30 (10%), MUM1 (40%), p53 (30%), and Ki67 (from 40% to 90%). Table 3 summarizes the immunohistochemical findings in DLBCL.
Aberrant phenotypes are not uncommon is DLBCL, and may be responsible for confusion in the diagnosis (Table 4). DLBCL may lack 1 or more B-cell markers such as CD19, CD20, CD22, and CD79a. DBCL may coexpression MUM1 and BCL6 (unlike normal germinal centers), or be positive for BCL6+ in the absence of t(14;18). Expression of T-cell markers is characteristically negative, although CD5+ and CD3+ variants of DLBCL have been described and are discussed below. DLBCL rarely express cyclin D1. In cyclin D1 positive cases there may be extra copies of cyclin D1 owing to trisomy of CCND1/chromosome 11.11 The blastoid variant of mantle cell lymphoma should be of course considered in the differential diagnosis of these cases.
MOLECULAR AND CYTOGENETICS OF DIFFUSE LARGE B-CELL LYMPHOMA
DLBCL is generally characterized by clonal rearrangements of immunoglobulin heavy and light chain genes. There are somatic hypermutations in the variable regions in most cases. These mutations may be ongoing in cases of germinal center-derived DLBCL. The most common genetic findings are abnormalities in 3q27 involving BCL6 in 30% of cases,12–14 and abnormalities of BCL2 t(14;18) in 20%, and MYC rearrangement in 10% of cases.15 Translocations t(14;18) and the MYC gene on chromosome 8 are associated with the germinal center derived-DLBL's,16 whereas BCL6 abnormalities are more common in cases with nongerminal center-derived lymphomas (Table 5).17
BONE MARROW INVOLVEMENT IN DIFFUSE LARGE B-CELL LYMPHOMA
Marrow involvement occurs in about 16% of cases of DLBCL. Two main patterns of bone marrow involvement occur with important clinical correlations.18–20 Bone marrow involvement may be concordant in which the cells infiltrating the marrow are large (large cell lymphoma), and this variant is generally associated with a poorer clinical outcome (5 y survival 10% in some series).21 In other cases the lymphoma cells infiltrating the marrow are small and occur in aggregates usually in paratrabecular location, resembling a low-grade lymphoma (so-called discordant histology).22,23 This pattern of marrow infiltration is more common and has been associated with more frequent relapse but a similar 5 year survival to cases without bone marrow involvement. Although DLBCL may form readily identifiable aggregates in the marrow, in some cases there is single cell infiltration which can be overlooked in routine hematoxylin and eosin sections, indicating the need for ancillary studies such as immunostains for CD20 (or other markers in rituximab treated cases) to rule out involvement in staging bone marrows24 (Figs. 1B, C). In about 30% of patients with bone marrow disease, circulating cells in the peripheral blood can be identified with flow cytometry.
CLASSIFICATION OF DIFFUSE LARGE B-CELL LYMPHOMA
The WHO 2008 classification has significantly expanded the spectrum of aggressive B-cell lymphoma subtypes, which are recognized as distinct or putative entities.7 DLBCL can be classified as DLBCL not otherwise specified (NOS), DLBCL subtypes, and other lymphomas of large B-cells (Table 2). The classification also recognizes borderline cases with overlapping features between BL and Hodgkin lymphoma, and these entities will be discussed in turn.
DIFFUSE LARGE B-CELL LYMPHOMA NOT OTHERWISE SPECIFIED
Morphologic subtypes recognizable with adequate histologic sections or cytologic preparations include centroblastic, immunoblastic, and anaplastic variants.
In the centroblastic variant of DLBCL the cells resemble the large centroblasts seen in the dark zone of germinal centers25 (Fig. 1D). The cells are large with round or oval nuclei, vesicular chromatin, and 2 to 4 nucleoli often evenly spaced and oriented at the nuclear membrane. These cells have moderate amphophilic to basophilic cytoplasm; a feature that may be missed in inadequately fixed specimens. There may be a uniform population of centroblasts, or there may be admixed a minor component of immunoblasts. In some cases, particularly in DLBCL primary in the bone or mediastinum, the nuclei may be lobated.
In the immunoblastic variant greater than 90% of the cells have an immunoblastic appearance with large centrally located nucleolus, basophilic or amphophilic cytoplasm, and sometimes paranuclear clearing (Fig. 1E).26 The anaplastic variant of DLBCL is characterized by large round oval or polygonal cells with bizarre pleomorphic nuclei27 (Fig. 1F). These cells may resemble Hodgkin cells and can have a sinusoidal or cohesive growth pattern resembling anaplastic large cell lymphoma. In addition there are rare cases of DLBCL in which the large neoplastic lymphoid cells have original cytologic features including spindle cells, cells with microvillous projections, or cytoplasmic vacuoles resembling signet ring cells of adenocarcinoma (Fig. 1G).
MOLECULAR SUBGROUPS OF DIFFUSE LARGE B-CELL LYMPHOMA NOT OTHERWISE SPECIFIED
On the basis of the gene expression profiling DLBCL can be characterized into at least 2 groups, germinal center B-cell (GCB) with signature of GCBs (50% cases), and nongerminal center including lymphomas with the signature of ABCs.28–33 Cases with the germinal center phenotype are less aggressive with significantly improved overall survival (approximately 60% overall 5 y survival compared with 35% for the ABC group).34,35 Cases with the ABC profile have gene expression similar to those induced by in vitro activation of peripheral blood, and this group has been associated with an adverse prognosis. Immunohistochemical markers for the nongerminal center group include Mum-1 (IRF4) and CD138.36
IMMUNOHISTOCHEMICAL SUBGROUPS OF DIFFUSE LARGE B-CELL LYMPHOMA NOT OTHERWISE SPECIFIED
A number of investigators have attempted to recapitulate the molecular subgroups described above and use immunohistochemical expression patterns as prognostic indicators in DLBCL.37–45 One of the most widely accepted has become known as the Hans classifier of DLBCL42 (Table 6). According to this scheme cases that are CD10+ in more than 30% of the cells are in the GCB group (Fig. 1H). Cases that are CD10 negative and BCL6+, MUM1 negative are also in the GCB group. Cases that are BCL6 negative, CD10 negative, or BCL6 positive and MUM1 positive are in the non-GCB group (Fig. 2). Expression of LMO2 correlates with the more favorable germinal center group,46 whereas FOXP1 expression is associated with an adverse prognosis and non-GCB signature. Expression of BCL2 protein is seen in the non-GCB group and correlates with an adverse prognosis even in the rituximab era.47–51 Mutations in p53 and high Ki67 scores have also been reported as independent poor prognostic variables.52–56
The CD5+ DLBCL is an unusual immunohistochemical subtype of DLBCL recognized by the WHO (Fig. 3). This lymphoma is characterized by a higher age at presentation (mean 66 y), female predominance, and advanced stage at diagnosis, “B” symptoms, and a more aggressive clinical course.57 It is thought that this subtype may arise from the same progenitor cell as the mutated variant of CD5+ SLL/CLL cell but these cases arise de novo without prior history of CLL or mantle cell lymphoma.58
In addition to the prognostic information provided by the GBC and ABC subgroups of DLBCL, expression of BCL2 protein is also associated with a worse prognosis even in the rituximab era.59 Immunohistochemistry may also help to identify other unique variants such as plasmablastic lymphomas which have a plasma cell phenotype, and are often negative for Pax5 and positive for CD138. Although translocations involving C-MYC are the hallmark of BLs,60 they can also occur in aggressive variants of DLBCL which can have rearrangements of MYC, BCL2, and BCL6.60,61
DIFFUSE LARGE B-CELL LYMPHOMA SUBTYPES
The following subtypes of DLBCL are recognized by the WHO 2008 and will be discussed:
(a) T-cell/histiocyte-rich LBCL,
(b) primary DLBCL of the central nervous system (CNS),
(c) primary cutaneous DLBCL Leg type, and
(d) EBV-positive DLBCL of the elderly.
TCRBCL is a morphologic variant in which there is a minor component of large malignant B-cells in a T-cell/histiocyte rich background. The presence of histiocytes in addition to lymphocytes is the characteristic feature of this lesion, and a CD68 or CD163 stain is often useful to highlight this component of the reactive background population. Large cells may be RS-like, centroblast/immunoblast like, or resemble “popcorn” cells seen in lymphocyte predominant Hodgkin lymphoma62–64 (Fig. 4). This variant may be confused with Hodgkin lymphoma particularly lymphocyte predominant Hodgkin lymphoma or lymphocyte rich classical Hodgkin lymphoma (CHL).65,66 TCRBCL is most closely related to the diffuse variant of lymphocyte predominance Hodgkin lymphoma from which it may be indistinguishable.
Clinical Features of T-cell Rich B-cell Lymphoma
This is an unusual morphologic variant counting for about 10% of cases of DLBCL. There is a broad age range from 12 to 61 years, and the disease predominantly affects males (from 3–4 to 1 ratio). TCRBCL usually presents with advanced stage (stage III or IV) and involves lymph nodes. Bone marrow, liver, and spleen are involved at diagnosis in up to 60% of cases. The disease is often refractory to therapy,67 although some studies have reported a similar outcome to DLBLC after combination chemotherapy.68
Histology Features of T-cell Rich B-cell Lymphoma
There is diffuse effacement of nodal architecture with tumor cells evenly dispersed within the clusters of bland histiocytes and small T-cells. Eosinophils or plasma cells are characteristically absent. The large cells have large vesicular nuclei and variably prominent nucleoli. Immunohistochemical stains are positive for pan B-cell markers such as CD20 and PAX5, and also express BCL6 (Fig. 5). Unlike CHL they are negative for CD15, CD30, and also negative for EBV. Molecular studies reveal clonally rearranged immunoglobulin genes carrying somatic mutations, similar to GCBs.69
PRIMARY DIFFUSE LARGE B-CELL LYMPHOMA OF THE CENTRAL NERVOUS SYSTEM
This subtype of DLBCL is rare (less than 1% of non-Hodgkin lymphomas), and defined as primary intracerebral or intraocular lymphoma. Primary CNS lymphoma has sufficiently distinct clinical and pathology features to be recognized as a distinct entity by the WHO.70,71 Lymphomas of the dura, intravascular lymphoma, lymphomas secondarily involving the CNS, and immunodeficiency lymphomas are excluded from this group. Primary CNS lymphoma is most common in older males >60. They may present as single space occupying lesions in the brain, although multiple lesions can also occur in about 30% of patients.
Pathology of Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System
Tumor cells mostly resemble large centroblasts or immunoblasts, and characteristically involve perivascular spaces (Fig. 6). They are thought to be derived from GCBs with aberrant somatic hypermutations.72,73 The malignant cells may occur in sheets or it can be intermixed with reactive small lymphocytes, macrophages, active microglial cells, and reactive astrocytes. Necrosis is frequently present. By immunohistochemical staining they are positive for pan B-cell markers, and frequently express BCL6 and MUM1.
PRIMARY CUTANEOUS B-CELL LYMPHOMA LEG TYPE
This entity is unusual in being defined by its site of most common presentation, and has distinct clinical and pathologic features. First recognized by Willemze et al and reported in the WHO-EORTC (World Health Organization European Organization for Research and Treatment of Cancer) classification of cutaneous lymphomas, the entity has been accepted by the WHO in 2008.74 This subtype of DLBCL occurs mostly in elderly females, mostly but not always on the lower leg.75 It is characterized histologically by sheets of centroblasts and immunoblasts and frequent mitoses (Fig. 7). In addition to pan B-cell markers these lymphomas characteristically express Bcl-2+ (unlike cutaneous follicular lymphoma),76 Bcl6+, Mum1/IRF4+, and are negative CD10 (Fig. 8). Molecular studies such as gene expression profiling and comparative genomic hybridization support reveal distinct features for the leg type of DLBCL compared with other cutaneous lymphomas.77 The absence of large cleaved cells (large centrocytes) is important in differentiating this entity from the more indolent cases of primary cutaneous follicular lymphoma. This is an aggressive form of DLBCL with only a 50% 5-year survival.78
EPSTEIN-BARR Virus -POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA OF THE ELDERLY
First recognized in the Japanese population, this subtype was previously known as ‘senile associated’-lymphoproliferative disorder, and is defined as a clonal EBV+ large cell lymphoma in patients with no known cause for immunodeficiency or prior lymphoma.79,80 The pathogenesis is thought to relate to senescence of the immune system.6,81,82 It is generally seen in those over 50 (but can occur in younger individuals) and the median age is 70 years. When making the diagnosis it is important to rule out other immunodeficiency syndromes, and exclude other EBV related lymphoproliferative disorders such as lymphogranulomatosis, mononucleosis, and primary effusion lymphomas (PEL). EBV should be localized in the malignant cells not bystander lymphocytes, and the characteristic pattern of viral latency in the large malignant cells is Epstein-Barr encoded RNA (EBER)+, latent membrane protein 1 (LMP1)+, EBNA2+.
Common to many EBV-related lymphoproliferative disorders the histology may vary over a spectrum from polymorphous proliferation resembling posttransplant lymphoproliferative disorder to monomorphous DLBCL. Geographical necrosis and large polyploid RS-like cells are common encountered (Fig. 9). A variable component of reactive lymphocytes/histiocytes may be present in the background again causing Hodgkin lymphoma to be considered in the differential diagnosis.
Immunophenotypic studies are helpful in the differential since most cases are CD20+, CD79a+, MUM1+, CD10−, BCL6− (Fig. 10). When present the H-RS cells are characteristically EBV+ (LMP1 and EBV nuclear antigen), CD20+, CD30+ in 75% of cases, and CD15 is negative. These lymphomas are monoclonal by molecular genetics and that is also helpful in distinguishing the polymorphous proliferations from infectious mononucleosis of the elderly.
OTHER LYMPHOMAS OF LARGE B-CELLS
The WHO 2008 has grouped a number of DLBCL within the spectrum of DLBCL in this category and each will be discussed with the exception of human herpesvirus8 (HHV8)-related lymphomas which are not included in this review:
* Primary mediastinal (thymic) large B-cell lymphoma (LBCL),
* Intravascular LBCL,
* DLBCL-associated with chronic inflammation,
* Lymphomatoid granulomatosis,
* ALK-positive LBCL,
* Plasmablastic lymphoma,
* Large B-cell lymphoma arising in HHV8+ multicentric Castleman disease*
* Primary Effusion Lymphoma*
PRIMARY MEDIASTINAL (THYMIC) LARGE B-cell LYMPHOMA
This entity is defined as DLBCL arising in the mediastinum from thymic B-cells which are normally present in this location.83 It comprises only 2% to 4% non-Hodgkin lymphoma, and occurs in young adults (median 35 y), with a female predominance. Clinically there may be a large mediastinal mass and patients can present with dyspnea and superior vena cava obstruction. Most patients have stage I/II disease, and marrow involvement is rare (3%). The disease is generally aggressive, but prognosis is now more favorable than previously thought owing to treatment with intensive chemo +/− radiation therapy (complete response rate 80%, plateau beyond 2 y). When cases relapse they may do so in unusual sites (GIT, kidney, adrenal, and CNS).84
Pathologic Features Primary Mediastinal Large B-cell Lymphoma
DLBCL of the mediastinum has a characteristic histologic appearance but these features are not required for diagnosis. Features usually present include sheets of large B-cells with clear cytoplasm and round, oval, or multilobated nuclei (Fig. 11). Sclerosis is commonly present, either broad bands of fibrous tissue, compartmentalizing sclerosis with fine collagen bands, or interstitial fibrous tissue. The immunophenotype is also distinctive, with the cells being immunoglobulin negative and lacking surface or cytoplasmic immunoglobulin. CD30 is positive in 80% cases, but staining is usually weak and heterogeneous.85 Seventy percent of cases are CD23+, and MAL is positive in 70%.86,87 Molecular studies reveal immunoglobulin gene rearrangement with somatic hypermutations. These lymphomas lack BCL2, BCL6, or MYC rearrangements.
INTRAVASCULAR LARGE B-CELL LYMPHOMA
Also known as “angiotropic large cell lymphoma”, intravascular lymphoma is characterized as large cell lymphoma with multifocal, selective growth within lumina of capillaries88,89 (Fig. 12). There are usually no circulating lymphoma cells. Any organ may be affected, and the disease is usually disseminated involving the marrow, CNS, and skin etc. Lymph nodes are usually uninvolved.
Clinical Features of Intravascular Lymphoma
The average age at presentation is 65 years although there is a broad range (from 34 to 85), and a slight male predominance. Patients present with weight loss, fever of unknown origin, malaise, and signs and symptoms related to occlusion of small vessels in various organs, most often the CNS and skin. There is an isolated cutaneous variant with a relatively good prognosis more common in females. Skin lesions include tender erythematous nodules, tumors, telangiectasia, cellulitis, lymphedema. Neurologic conditions are highly variable depending on the location of the infiltrates, and include infarcts, dementia, polyneuropathy, and myalgia.
Pathologic Features of Intravascular Lymphoma
Because of the intravascular localization of the malignant cells without the formation of tumor masses it is easy to overlook intravascular lymphoma particularly in a bone marrow biopsy (Fig. 13). Also the diagnosis should only be made when the lymphoma is localized to intravascular spaces as secondary vascular involvement may be seen in many lymphomas. The cells are usually large with prominent nucleoli, and may be accompanied by fibrin thrombi, hemorrhage, and necrosis. From 85% to 91% of cases are B-cells expressing the usual pan-B cell markers, but there are rare T-cell and NK cell variants.90 The cells are Bcl-2+, CD43+, and there is variable expression of CD5, CD10, and MUM1. From 20% to 25% of cases express BCL-6, whereas CD23 and cyclin D1 are negative. There is an Asian variant which is usually positive for CD5.90,91
DLBCL-ASSOCIATED WITH CHRONIC INFLAMMATION
DLBCL-associated with chronic inflammation was previously known as pyothorax-associated lymphoma.92–96 This is an extremely rare form of DLBCL occurring in association with long standing chronic inflammation mostly involving body cavities. There is an association with EBV, and the lymphomas must be distinguished from Kaposi sarcoma associated herpesvirus positive PEL. Morphologic features in tumor masses are similar to DLBCL NOS, and most cases show centroblastic or immunoblastic morphology. This is an aggressive form of lymphoma with an overall survival of less than 35%.
Lymphomatoid granulomatosis is an angiocentric angiodestructive lymphoproliferative disease involving extranodal sites, composed of EBV+ B-cells admixed with reactive T-cells which usually predominate.97–101 It is more common in adult males, and characteristically involves the lungs with bilateral peripheral lung nodules, sometimes a solitary mass, or diffuse infiltration. Other sites of involvement include skin, CNS, kidney, and liver. It is important to correctly diagnosis and categorizes this entity as the early lesions (grade 1 or 2) may wax and wane or respond to interferon α. Grade 3 lesions are more aggressive usually requiring chemotherapy regimens with rituximab. Table 7 summarizes the criteria for prognostic grading of cases of lymphomatoid granulomatosis on the basis of number of large EBV positive cells and other features.
Pathology of Lymphomatoid Granulomatosis
Pulmonary nodules vary in size and are often multiple or bilateral. There is a histologic triad for making the diagnosis, which includes an atypical polymorphous lymphoid infiltrate often with a granuloma-like appearance, an angiocentric and angiodestructive infiltrate, and prominent geographic necrosis (Fig. 14). Immunohistochemical studies are helpful in confirming the diagnosis with the presence of CD20+ large cells and CD3+ reactive small T-cells and histiocytes in the background (Fig. 15). In situ hybridization for EBER and immunostain for EBV LMP1 highlight the abnormal B-cell population within the inflammatory infiltrate. Molecular studies may be variable but most of the higher-grade lesions have immunoglobulin gene rearrangements.
ANAPLASTIC LYMPHOMA KINASE-POSITIVE LARGE B-CELL LYMPHOMA
ALK-positive LBCL is a B-cell lymphoma with sheets of monomorphic large immunoblast-like B-cells, sometimes with plasmablastic differentiation. The cells express ALK kinase but generally lack the characteristic 2;5 translocation seen in T/NK cell anaplastic large cell lymphoma.102,103 This lymphoma seems more common in adult males but occurs in all ages including children. This is a rare lymphoma and cases have been reported at both nodal and extranodal locations.
Pathology of Anaplastic Lymphoma Kinase-positive large B-cell lymphoma
The lymphoma consists of immunoblast or plasmablastic cells and may have a sinusoidal location. Multinucleated giant cells may also be present. Malignant cells may be positive for CD79a and usually express EMA and plasma cell markers including CD138. CD20 is usually negative and CD45 is weak or absent. CD30 staining is weak or negative. The most characteristic finding is granular cytoplasmic staining for ALK protein (Fig. 16). In most cases this corresponds to the t(2;17) translocation responsible for the clathrin-ALK fusion protein.104,105
Plasmablastic lymphoma is described as a diffuse proliferation of large cells which resemble B-immunoblasts or plasmablasts, but have the phenotype of plasma cells.106–116 The entity seems heterogeneous, and includes cases of the oral mucosa type discussed below. Other cases with more overt plasmacytic differentiation seem closer to the blastic form of plasmacytoma, and clinical features may be required to differentiate these cases from myeloma.117
Plasmablastic Lymphoma of the Oral Mucosa Type
First described in the oral cavity of patients with human immunodeficiency virus (HIV), it is now appreciated that there is a wider distribution of this lymphoma and that it also occurs in other, albeit predominantly extranodal sites. Although the highest association is with HIV, plasmablastic lymphoma can occur with other immunodeficiency including the elderly. In HIV+ patients there is a predilection for involvement of the oral cavity, nasal, and paranasal areas. Nodal involvement is more common in cases not associated with HIV, and these cases may have a more plasmacytic appearance. In some cases there is no history of immunodeficiency. The majority is EBV+, but they are negative for Kaposi sarcoma associated herpesvirus. The differential includes HHV-8-associated lymphomas (PEL, extracavitary PEL, multicentric Castleman's), and plasmablastic myeloma. If the lymphoma is HHV-8+ (usually identified with the LANA stain in paraffin sections) the lymphoma should be considered a form of PEL and not plasmablastic lymphoma. Most patients are stage IV and the disease has an aggressive clinical course.
Pathologic Features Plasmablastic Lymphoma of the Oral Cavity Type
The morphologic spectrum of this disease may make the diagnosis difficult without reference to clinical history and immunophenotypic characteristics. There is a diffuse proliferation of cells which appear cohesive resemble immunoblasts (Fig. 17). The immunophenotype is characteristic with cells staining for CD138 and CD38 and often EMA and Mum1. Expression of CD45 and pan B-cell markers CD20 and Pax 5 is weak. CD79a is positive in 50% to 85% cases. Many cases express cytoplasmic immunoglobulin, usually IgG. EBV-EBER is positive in up to 75% of cases in the oral cavity type.
Plasmablastic Lymphomas With Overt Plasmacytic Differentiation
These cases may be difficult to distinguish from plasmacytoma/myeloma and clinical information may be required for complete evaluation. CD56 is usually absent in the lymphomas, and when present should raise the suspicion for plasmablastic myeloma.
BL is described as a monomorphic proliferation of medium sized transformed B-cells with round nuclei, clumped chromatin, basophilic cytoplasm, squared off cell borders, cytoplasmic vacuoles, medium sized paracentral nucleoli, and a starry sky pattern. Translocation involving MYC characteristic but not specific for BL or always identified. No single parameter is the gold standard for diagnosis; morphology, cytogenetics, immunophenotype, and expression profiles all may contribute to the diagnosis.59,118–124 Whereas there may be some morphologic variation in the cellular composition of BL, this finding alone does not obviate the diagnosis, and the confusing terms of Burkitt-like or atypical BL have been eliminated by the WHO and should no longer be used.
Burkitt Lymphoma Clinical Features
BLs comprises 1% to 2% of the lymphomas in the United States America, but about 30% to 50% of childhood lymphomas. The median age in the adult group is 30 years. In the West the majority present as abdominal masses with ileocecal and other organ involvement. They may also present as acute leukemias. Bone marrow involvement at presentation is uncommon with the exception of HIV-associated BL. BL is usually treated with aggressive therapy (hyper cyclophosphamide, vincristine, adriamycin, and dexamethasone or related regimen plus CNS prophylaxis). There are 3 clinical variants of BL.
Endemic BL usually occurs in equatorial Africa and is associated with EBV in almost all cases. This variant usually occurs in children with tumor masses in the jaws, gonads, and kidneys.
Sporadic BL is encountered in the West in immunocompetent patients, mainly children and young adults. EBV is positive in about 40% of the cases. This variant commonly involves the terminal ileum and Waldeyer's ring.
AIDS-associated BL occurs in patients infected with HIV. Most patients have relatively high CD4 counts compared with other non-Hodgkin lymphomas. There is frequent nodal and bone marrow localization.125–127
Pathology of Burkitt Lymphoma
BL is characterized by sheets of intermediate sized cells, which are often cohesive with squared off borders. Nucleoli are small and may be multiple. The cytoplasm is basophilic and contains lipid vacuoles best appreciated on imprints and smears (Fig. 18). This feature is characteristic but no specific for BL cells. Because of the rapid proliferation rate there are numerous mitoses, as well as tingible body or ‘starry sky’ macrophages, which phagocytose the apoptotic debris. The neoplastic cells have a germinal center phenotype expressing IgM+, CD10+, BCL2−, TdT−, BCL6+, and Ki67 approaching 100% (Fig. 19). There are few reactive CD3+ T-cells. The cells are IgM+, and the immunoglobulin genes are hyper mutated but there is no class switch. BLs commonly express TCL1 which is not specific for Burkitt but may be associated with the MYC translocation.128,129 Documentation of MYC translocation is highly desirable but no essential for diagnosis.
Although the diagnosis of BL is usually easy when the characteristic features are present, it may be difficult in cases which are not entirely typical either because of morphologic or immunophenotypic overlap with other lymphomas. Using gene expression profiling as the gold standard even expert pathologists may have difficult in differentiating between BL and other high-grade lymphoma.130–134 For example BL may be negative for BCL6, particularly in EBV positive cases in which the BCL6 is down-regulated. Although the absence of BCL2 is characteristic of BL, a subset may be weakly positive. Although BL cases are characteristically uniform in their cellular makeup there may be more variation in some cases, particularly following therapy. In AIDS-related BLs the neoplastic cells typically have a more plasmacytoid cytoplasmic appearance (Fig. 20). The term atypical BL was used to classify cases of BL cells with more pronounced variation in size and shape and/or large central nucleoli. This was a confusing category eliminated by WHO 2008. There are cases with overlapping clinical features between BL and DLBCL and these are discussed below.130
The MYC translocation is characteristic of BL but can be seen in other non-Hodgkin lymphomas including so-called ‘double-hit’ lymphomas which have both t(14;18) and MYC translocations. The translocation in BL involves the MYC gene on chromosome 8, and either the heavy chain, κ, or λ light chain genes on chromosomes 14, 2, and 22, respectively. The MYC break apart probe is usually used in interphase fluorescent in situ hybridization to identify MYC translocations since it is not dependent on the specific translocation partner. Not all cases are positive with the interphase fluorescent in situ hybridization probe, however, as the translocation can involve other partners, which may be missed without standard cytogenetics. In BL the karyotype is usually simple, unlike other high-grade non-Hodgkin lymphomas which have MYC translocations in addition to a complex karyotype.
B-CELL LYMPHOMA, UNCLASSIFIABLE, WITH FEATURES INTERMEDIATE BETWEEN DIFFUSE LARGE B-CELL LYMPHOMA AND BURKITT LYMPHOMA (BCLUWFIBDLBCLABL)
This terminology was adopted by WHO 2008 for aggressive B-cell lymphomas with morphologic, phenotypic, and genetic features, which overlap between BL and DLBCL. Formerly these would usually been in the unclassified category or classified as Burkitt-like lymphomas. This is not a distinct entity in the WHO but is useful for classifying cases, which do not meet the diagnostic criteria for BL or DLBCL. Some cases of transformed follicular lymphoma will also fall into this category. Many cases resemble BL in terms of morphologic features and phenotype, but have one or more features that preclude that diagnosis. Most of “double hit” lymphomas, with both t(14;18) and translocations involving C-MYC, fall into this category and should be recognized because of their aggressive clinical behavior (Fig. 21). Although BL cases may show some morphologic variation (formerly called atypical BL), morphology alone is insufficient to place a case in this grey zone category.
This lymphoma usually occurs in adults some of whom have history of follicular lymphoma. Many patients present with generalized adenopathy or extranodal disease. The disease is aggressive with a poor clinical outcome.135–137
Included in this group are cases which morphologically resemble BL but have immunophenotypic or molecular features which are not compatible with this diagnosis.59,118,138 Some cases resemble DLBCL but features of BL including starry sky macrophages, rapid proliferation rate, and apoptotic bodies are usually present. Other cases are intermediate in size between Burkitt and DLBCL with irregular nuclear outlines and prominent nucleoli. “Blastoid” cases with fine granular chromatin and inconspicuous nucleoli but TdT and cyclin D1 negative are also included. Cases of DLBCL should not be included in this group solely because of rapid proliferation and/or MYC translocations.
Immunohistochemical and Molecular Features
Phenotypically the cells are positive for pan-B cell markers. Most express surface immunoglobulin. Many cases resemble BL in being CD10+, BCL6+, Ki67 high, MUM1−, but unlike BL they are often BCL2+.139 Cytogenetic studies usually reveal a complex karyotype unlike BL. Cases of “double-hit” lymphoma with t(14;18) or BCL6 and MYC translocations are also included.140 The “double-hit” cases may be negative for surface immunoglobulins. Only about 60% of cases with MYC translocations involve t(8;14), and in 40% of the cases there are other translocation partners.
B-CELL LYMPHOMA, UNCLASSIFIABLE, WITH FEATURES INTERMEDIATE BETWEEN DIFFUSE LARGE B-CELL LYMPHOMA AND CLASSICAL HODGKIN LYMPHOMA (BCLUWFIBDLBCLACHL)
Formerly called grey zone lymphoma,141 this is a B lineage lymphoma with overlapping clinical, morphological and/or immunophenotypic features with CHL and DLBCL, especially primary mediastinal large B-cell lymphoma (PMBL).141–144 PMBCL and CHL share common gene expression profile and both are thought to arise from a common precursor thymic B-cell.145,146 Interestingly PMBL is more common in females, whereas the grey zone lymphoma is most common in young men. Table 8 is a summary of the key features in the differential diagnosis between CHL and PMBL.
Typically grey zone lymphomas occur in the mediastinum of young men between ages from 20 to 40, and may be positive for CD20 but also for CD15. They have an aggressive clinical course. Involvement of adjacent lymph nodes in the supraclavicular area is common, and there may be spread to the lung by direct extension. Liver, spleen, and bone marrow may also be involved. Less commonly these lymphomas have been reported at other nodal sites. Treatment is controversial but there are new treatment regimens such as etoposide, adriamycin, vincristine, cyclophosphamide, prednisone and rituximab currently in clinical trials to improve the outcome.
Many of these cases resemble CHL but have abnormal features such as a large number of CD20+, CD45+ mononuclear cells, and diminished background inflammatory cells and sclerosis. Some of these lymphomas may present as CHL and subsequently develop PMBCL. Composite CHL and PMBCL are also related. Alternatively these cases may resemble mediastinal LBCL but contain RS cells and an aberrant phenotype such as CD20−, CD15+ (Figs. 22A, B). The phenotype is often intermediate between CHL and DLBCL, with neoplastic cells CD45+, CD30+, Pax5+, CD20+/− CD79a+/−, CD10−, Bcl6−/+, Oct2+/−, Bob1+/−, CD15+/−. Pan B-cell markers are usually expressed by the majority of the tumor cells. The cells usually lack surface and cytoplasmic immunoglobulin. In cases, which resemble PMBL, expression of CD15 or EBV favors the diagnosis of BCLUWFIBDLBCLACHL. MAL may be expressed in a subset of cases.
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