B-Cell Lymphoma Workup

The initial evaluation of the patient with newly diagnosed non-Hodgkin lymphoma (NHL) must establish the precise histologic subtype, the extent and sites of disease (localized or advanced; nodal or extranodal), and the patient's general health status. These studies help to determine the appropriate therapy and the patient's prognosis.

Tissue Biopsy

Incisional or excisional biopsy is preferred for the initial diagnosis; core needle biopsy is discouraged except for cases in which a lymph node is not easily accessible. Fine-needle aspiration is not an acceptable diagnostic tool; however, in selected circumstances it can be used in combination with immunohistochemistry and flow cytometry.

Flow cytometry and immunohistochemical stains of the biopsied material should be performed to confirm the diagnosis and for accurate subtyping

Laboratory studies are as follows:

• Complete blood cell count (CBC) with differential and examination of a peripheral smear – To assess bone marrow function and rule out the presence of abnormal circulating cells in the peripheral blood
• Screening chemistries – To assess renal and hepatic function and measure serum glucose, calcium, albumin, lactate dehydrogenase (LDH), and beta2-microglobulin
• Serum protein electrophoresis – Frequently appropriate
• HIV serology – Appropriate for patients with risk factors, especially those with large cell or small noncleaved-cell histologies

Immunoglobulin gene rearrangement

Immunoglobulin (Ig) gene rearrangement is useful for differentiating a B-cell lymphoproliferative process from a monoclonal or reactive proliferation of lymphocytes. This technique not only provides a specific marker for B cells but also is a true marker for monoclonality.

Polymerase chain reaction

Polymerase chain reaction (PCR) is used to assess minimal residual disease. This technique has generally been applied to the t(14;18) translocation and the associated bcl-2 gene.

Whereas PCR positivity for bcl-2 gene rearrangements can be found in healthy individuals, patients with lymphoma in remission who show positive results for bcl-2 gene rearrangements by PCR in the blood or bone marrow are more likely to experience relapse than patients who do not have this abnormality. However, the correlation between relapse and PCR positivity is not perfect, and, at present, PCR is still considered a research tool.

Fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET/CT) is the preferred imaging modality for staging FDG-avid nodal lymphomas, while CT alone is preferred for FDG-nonavid and variably FDG-avid histologies. ^[1]

For these purposes, essentially all nodal lymphoma histologies are considered FDG-avid except the following:

• Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL)
• Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia
• Mycosis fungoides
• Marginal zone lymphoma

Although PET/CT is generally not useful for staging these more indolent histologies, it may be helpful in some circumstances (eg, to identify a preferred biopsy site if aggressive transformation is suspected).  

A dedicated contrast-enhanced CT scan may be required in addition to the PET/CT to define the extent of disease in special situations, such as in the setting of lymphadenopathy close to bowel or if there is compression or thrombosis of blood vessels. Additional tests may be helpful in the evaluation of specific disease sites. Specific evaluation of the gastrointestinal tract, liver, spleen, central nervous system, skeleton, or genitourinary tract is reserved for patients with symptoms or those at particularly high risk for involvement of these sites.

The histologic findings in B-cell non-Hodgkin lymphoma (NHL) are varied. The salient features of the most common subtypes are described below.

Chronic lymphocytic leukemia/small lymphocytic lymphoma

Chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) is a neoplasm composed of monomorphic small, round to slightly irregular B lymphocytes in the peripheral blood, bone marrow, spleen, and lymph nodes, admixed with prolymphocytes and paraimmunoblasts forming proliferation centers in tissue infiltrates.

The lymph node architecture is effaced, with a pseudofollicular pattern of regularly distributed pale areas corresponding to proliferation centers containing larger cells in a dark background of small cells. The predominant cell type is the small lymphocyte. Mitotic activity is usually low. The size of proliferation centers and the number of paraimmunoblasts vary from case to case, but there is no correlation between lymph node histology and clinical course.

On peripheral blood smears and bone marrow aspirate smears the CLL/SLL cells are small lymphocytes with clumped chromatin and scant cytoplasm. Smudge or basket cells are typically seen in peripheral blood smears.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue

Mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal lymphoma composed of morphologically heterogeneous small B cells, including marginal zone (centrocytelike) cells, small lymphocytes, and scattered immunoblast and centroblastlike cells.

The lymphoma cells infiltrate around reactive B-cell follicles, external to a preserved follicle mantle, in a marginal zone distribution and spread out to form larger confluent areas that eventually overrun some or most of the follicles. There is plasma cell differentiation in a proportion of the cases. In epithelial tissues, the neoplastic cells typically infiltrate the epithelium, forming lymphoepithelial lesions.

Nodal marginal zone lymphoma

Nodal marginal zone lymphoma (NMZL) is a primary nodal B-cell neoplasm that morphologically resembles lymph nodes involved by marginal zone lymphoma of extranodal or splenic types, but without evidence of extranodal or splenic disease.

Follicular lymphoma

Follicular lymphoma is a neoplasm composed of follicle center B cells (typically both centrocytes and centroblasts) that usually has at least a partially follicular pattern. It is graded by counting or estimating the absolute number of centroblasts in 10 neoplastic follicles, expressed per 40× high-power microscopic field (hpf). Grade 1 and grade 2 cases have a marked predominance of centrocytes and only a few centroblasts (grade 1, 0-5 centroblasts/hpf; grade 2, 6-15 centroblasts/hpf; grade 3, >15 centroblasts/hpf).

If diffuse areas of any size composed predominantly or entirely of blastic cells are present in any case of follicular lymphoma, a diagnosis of diffuse large B-cell lymphoma (DLBCL) is also made. Peripheral blood involvement sufficient to produce lymphocytosis (usually < 20,000/µL) is observed in about 10% of patients. Bone marrow involvement occurs in 65% of patients and characteristically takes the form of paratrabecular lymphoid aggregates. Splenic white pulp and hepatic portal triads are also frequently involved.

Mantle cell lymphoma

Mantle cell lymphoma is a B-cell neoplasm generally composed of monomorphic small to medium-sized lymphoid cells with irregular nuclear contours and a cyclin-D1 translocation (see the image below). Neoplastic transformed cells resembling centroblasts, immunoblasts, or paraimmunoblasts and proliferation centers are absent. Hyalinized small vessels are commonly seen.

A spectrum of morphologic variants is recognized. The blastoid and pleomorphic variants are considered to have poorer prognosis and to be of important clinical significance. Further evaluation of the proliferation fraction, either by counting mitotic figures or estimating the proportion of Ki67-positive nuclei, is important because of its prognostic impact.

Diffuse large B-cell lymphoma

The common morphologic features that unite the various forms of DLBCL are the relatively large cell size (usually 4-5 times that of a small lymphocyte) and a diffuse pattern of growth. ^[13] In other respects, a fair degree of morphologic variation exists.

In most cases, the tumor cells have a round or oval nucleus that appears vesicular because of margination of chromatin at the nuclear membrane, but large multilobed or cleaved nuclei predominate in some cases (see the image below). Nucleoli may be 2-3 in number and located adjacent to the nuclear membrane, or they may be single and centrally placed. Cytoplasm is usually present in moderate abundance and may be pale or basophilic.

Other more anaplastic tumors may contain multinucleated cells with large inclusionlike nucleoli that closely resemble Reed-Sternberg cells, and phenotyping is often necessary to distinguish these 2 entities.

An immunophenotypical subdivision of DLBCL has been proposed that uses a combination of antibodies to CD10, bcl-6, and MUM-1 to divide DLBCL into germinal center B cell-like (GCB) and nongerminal center B cell-like (non-GCB) varieties. Cases that are CD10 positive or CD10 negative and bcl-6 positive but MUM-1 negative are regarded as GCB type, whereas all others are regarded as non-GCB or activated B-cell (ABC) type.

This immunophenotypical subdivision does not completely correlate with gene expression-based subgrouping of DLBCL. In the non-GCB or the ABC, bcl-2 expression is known to be associated with poor patient outcome.

DLBCL may be classified in several different ways. Common morphologic variants of DLBCL, not otherwise specified, include centroblastic, immunoblastic, anaplastic, and rare morphologic variants. Molecular subgroups include GCB and ABC. Immunohistochemical subgroups include CD5-positive DLBCL, GCB, and non-GCB.

DLBCL subtypes include the following:

• T-cell/histiocyte rich large B-cell lymphoma
• Primary DLBCL of the central nervous system (CNS)
• Primary cutaneous DLBCL, leg
• Epstein-Barr virus (EBV)–positive DLBCL of the elderly

Other lymphomas of large B cells include the following:

• Primary mediastinal (thymic) large B-cell lymphoma
• Intravascular large B-cell lymphoma
• DLBCL associated with chronic inflammation
• Lymphomatoid granulomatosis
• Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma
• Plasmablastic lymphoma
• Large B-cell lymphoma arising in human herpesvirus 8 (HHV-8)–associated multicentric Castleman disease
• Primary effusion lymphoma

Borderline cases include the following:

• B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma
• B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin disease

Burkitt lymphoma

Burkitt lymphoma is a B-cell lymphoma with an extremely short doubling time that often presents in extranodal sites or as an acute leukemia. Involved tissues are effaced by a diffuse infiltrate of monomorphic, medium-sized (nuclei similar or smaller to those of histiocytes) transformed lymphoid cells.

The nuclei are round with finely clumped and dispersed chromatin, with multiple basophilic medium-sized paracentrically situated nucleoli. The cytoplasm is deeply basophilic and usually contains lipid vacuoles (see the images below). A high mitotic index is typical, as is apoptotic tumor cell death, accounting for the presence of numerous tissue macrophages with their ingested tissue debris. These macrophages are often surrounded by a clear space, creating the characteristic starry sky pattern.

Primary mediastinal (thymic) large B-cell lymphoma

Primary mediastinal large B-cell lymphoma is a diffuse large B-cell lymphoma that arises in the mediastinum from a putative thymic B-cell origin and has distinctive clinical, phenotypic, and genotypic features. Histologically, it is commonly associated with compartmentalizing alveolar fibrosis (see the image below). The cells are medium-sized to large and have abundant clear/pale cytoplasm and round to oval nuclei. A few cases have multilobated and pleomorphic nuclei resembling the Reed-Sternberg cells of Hodgkin lymphoma.

Flow cytometry analysis can help provide accurate diagnosis of some categories of B-cell lymphoma. Characteristic immunophenotypes are associated with major types of lymphoma (see the table below).

Table 2. Characteristic Immunophenotypes of Major Subtypes of Lymphoma (Open Table in a new window)

The Ann Arbor staging system has commonly been used for patients with non-Hodgkin lymphoma (NHL). ^[14] This system divides NHL into four stages and subclassifies it into A and B categories. The B designation is applied to individuals with any of the following well-defined generalized symptoms:

• Unexplained loss of greater than 10% of body weight in the 6 months before diagnosis
• Unexplained fever with temperature higher than 38°C
• Drenching night sweats

In 2014, the International Conference on Malignant Lymphomas (a multidisciplinary team of researchers representing major lymphoma clinical trial groups and cancer centers from North America, Europe, Japan, and Australasia) met in Lugano, Italy and created a modification of the Ann Arbor system, the Lugano classification. ^[15] The 2015 NCCN Non-Hodgkin’s Lymphomas guidelines adopted the Lugano classification. ^[16]

The Lugano classification (see Table 3, below) includes four stages. However, the A and B subclassifications are not required in NHL since they are not prognostic.

Table 3. Lugano Modification of the Ann Arbor Staging System. (Open Table in a new window)

Other staging considerations

The following factors are not a part of the Ann Arbor staging system, but have important prognostic value in B-cell NHL:

• Age
• Performance status
• Tumor size
• Lactate dehydrogenase (LDH) values
• Number of extranodal sites involved

To identify subgroups of patients most likely to relapse, the International Prognostic Index (IPI) was compiled for 2031 patients with aggressive NHL. ^[9] The IPI has been validated by several cancer centers and has been incorporated into new trials designed by various cooperative groups. (See Prognosis.)