Approach Considerations
The initial evaluation of diffuse large B-cell lymphoma (DLBCL) patients is aimed at determining the stage of the disease, assessing for end-organ damage by the disease and/or preexisting comorbid conditions, and identifying situations that may affect treatment design. The evaluation of patients with lymphoproliferative disorders should include a detailed history and physical examination.
Complete blood cell (CBC) count with differential
Laboratory testing to assess bone marrow and immunological function includes CBC count with differential and platelet counts, which should be performed in all newly diagnosed patients to evaluate involvement of the bone marrow, which may result in anemia, thrombocytopenia, and/or leukopenia. In addition, perform a peripheral blood examination for circulating tumor cells by flow cytometry.
Comprehensive metabolic panel
A comprehensive chemistry panel should be performed to help evaluate serum electrolytes, lactic dehydrogenase (LDH), renal function, and hepatic function. In addition, perform serum β2 microglobulin (B-cell lymphoma) testing.
Electrolyte abnormalities may occur from renal involvement with lymphoma. Abnormal renal function may require a chemotherapy dose adjustment.
Elevated levels of lactate dehydrogenase (LDH) and uric acid correspond with the tumor burden. The LDH value is a factor in the International Prognostic Index (IPI) and is a useful indicator of the extent of disease and of the response to treatment. It can be used as an early, nonspecific indicator of disease relapse. [44] An elevated uric acid level also signifies an increased likelihood of tumor lysis syndrome with chemotherapy
Computed tomography (CT) scanning
Routine CT scanning of the neck, chest, abdomen, and pelvis is the standard imaging study for patients with lymphoma.
Functional imaging
Functional imaging with positron emission tomography (PET) has become an important diagnostic and prognostic tool in the management of patients with various subtypes of lymphoma, such as DLBCL, Hodgkin lymphoma (HL), and mantle cell lymphoma (MCL). Most academic institutions recommend routine PET scanning to complement staging and posttreatment evaluation of all patients with aggressive lymphoma. Retrospective studies have demonstrated that midtreatment and/or end-of-treatment PET scanning provides strong prognostic information in terms of progression-free survival (PFS) and overall survival (OS). The adequate timing for PET scanning for response assessment is a subject of controversy and recommendations vary among academic institutions. [45]
Bone marrow sampling
Bone marrow biopsy and aspiration should be performed in all newly diagnosed lymphoma patients. Routine pathological, flow cytometry, and cytogenetic studies should also be performed.
CNS imaging and lumbar puncture
CNS imaging and cerebrospinal (CSF) analysis should be considered in clinically symptomatic patients or in those patients at high risk for occult CNS disease (eg, high-grade lymphoma, HIV-associated lymphoma, select patients with aggressive lymphoma).
Gastrointestinal imaging
While gastrointestinal evaluation (ie, upper and/or lower endoscopy) is recommended in patients with MCL, it is not routinely required in the staging of DLBCL.
Cardiac evaluation
Cardiac studies evaluating ejection fraction are required in patients expected to receive anthracycline-based chemotherapy.
Serological evaluation for pathogens
This includes evaluations for hepatitis B virus (HBV), and hepatitis C virus (HCV), and HIV.
Serological evaluation for HBV and HCV is mandatory for those patients expected to receive monoclonal antibody–based therapy (ie, rituximab). As rituximab use has become an integral part of the management of DLBCL, rare but serious adverse events have been associated with hepatitis B and C reactivation. Fatal cases of fulminate hepatic failure due to active hepatitis B or C have been reported in DLBCL patients undergoing chemoimmunotherapy. Therefore, routine HBV and HCV serology is recommended.
In addition, serologic testing for HIV infection should be considered in patients with coexistent HIV risk factors, aggressive histologies (DLBCL, Burkitt or T-cell lymphoma), or unusual clinical presentations.
Flow Cytometry and Genetic Studies
Flow cytometry to identify the expression of different immunophenotypes helps in determining a clonal cell population and in differentiating between B- and T-cell origins.
Cytogenetic or fluorescent in situ hybridization (FISH) studies may reveal chromosomal translocations characteristic of diffuse large B-cell lymphoma (DLBCL), such as the following:
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t(3q27) (bcl-6) - Occurs in 35% of patients
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t(14;18)(q32;q21) (bcl-2) - Occurs in 15-20% of patients
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t(8;14)(q24;q32) - Occurs in less than 5% of patients
Gene expression profiling may be able to distinguish 2 separate subtypes of DLBCLs (normal germinal center B-cell pattern vs activated B-cell–like), each of which has a different prognosis. In addition, results from gene rearrangement studies can be used to establish clonality. [46, 47]
Imaging Studies
CT scanning
CT scans of the neck, chest, abdomen, and pelvis are used to help identify the degree of lymphadenopathy, the presence or absence of extranodal disease, and/or the presence of visceral involvement. CT scans are also part of the complete staging workup for diffuse large cell lymphoma.
In addition, baseline CT-scan findings aid in disease follow-up care after chemotherapy to assess the degree of response to therapy; they also aid in planning consolidating radiation therapy, if used. (See the images below.)
Computed tomography (CT) scan of the abdomen showing mesenteric and retroperitoneal adenopathy in a patient with diffuse large cell lymphoma.
Patient with diffuse large B-cell lymphoma with extranodal involvement. This computed tomography (CT) scan shows an enlarged spleen and liver as a result of lymphomatous involvement.
Patient with diffuse large B-cell lymphoma with extranodal involvement (same patient as in the previous image). This patient has an enlarged spleen and liver as a result of lymphomatous involvement. Extensive retroperitoneal lymphadenopathy is also present.
Diffuse large B-cell lymphoma. Hematoxylin and eosin stain of a lymph node biopsy sample showing a mixture of large and small cells. The architecture of the node is lost, with a diffuse pattern of involvement.
Bone imaging
Patients with unexplained bone pain or elevated alkaline phosphatase levels should be evaluated with a bone scan. Obtain plain radiographs of any abnormal area on the bone scan to check for lymphomatous involvement of the skeleton.
Gallium-67 scanning
Gallium-67 (67Ga) scans are valuable in the staging of diffuse large cell lymphomas (DLCLs). Gallium uptake correlates with disease activity and is useful as an indicator of response and prognosis. Uptake of 67Ga occurs in approximately 50% of indolent lymphomas and in most aggressive and highly aggressive types. 67Ga scans are also sometimes used as a means of assessing sites of relapse. (See the image below.)
This image depicts gallium scans performed as part of a staging workup for a patient with diffuse large B-cell lymphoma. The scans show extensive hepatosplenic and multiple sites of nodal involvement with a gallium-avid tumor.
MUGA scanning
Multigated acquisition (MUGA) scans are used to evaluate the patient's ejection fraction before chemotherapy, because anthracyclines used in the treatment of diffuse large cell lymphomas have a potential cardiotoxic effect.
PET scanning
Positron emission tomography (PET) scans are increasingly being used to stage disease through the use of fluorodeoxyglucose (FDG), since findings for glucose uptake can indicate areas of increased metabolic activity. [48] PET-scan data can also be useful in helping to determine whether residual masses represent scars or persistent lymphoma.
PET scan findings are being investigated for use as prognostic indicators during treatment (after 2-4 cycles), but the clinical utility of this is still unclear.
PET scanning may be more sensitive than gallium scans for more indolent lymphoproliferative diseases, but definitive data comparing gallium to PET scanning of lymphomas are not available.
Biopsy and Lumbar Puncture
Bone marrow aspiration and biopsy is performed as part of the staging process to help rule out involvement with lymphoma. Bilateral iliac crest bone marrow biopsies should also be performed as part of the staging.
Lymph node biopsy is required to establish a definitive diagnosis of non-Hodgkin lymphoma (NHL); this should be an excisional biopsy rather than a needle biopsy, because nodal architecture is often difficult to assess when small amounts of tissue are used.
Because bone marrow involvement increases the likelihood of lymphomatous involvement of the meninges, in patients with advanced-stage disease, a lumbar puncture for cytologic and chemical analysis of the cerebrospinal fluid may be necessary.
Histologic Findings
The diagnosis of diffuse large cell lymphoma is usually confirmed after positive findings are obtained from a lymph node biopsy specimen. Pathology findings should be reviewed by an expert hematopathologist, because lymphomas can be difficult to classify.
Diffuse large B-cell lymphomas are more or less composed of equal numbers of small and large cells. The small cells are usually slightly larger than normal lymphocytes, and they have a cleaved or indented nucleus and coarse chromatin.
The large cells can be cleaved or noncleaved. The cytoplasm of these cells is pale, and the cells have an irregular, central, indented nucleus with inconspicuous nucleoli. A subset of the large cells has rounded nuclei with 1 or more nucleoli; these are the noncleaved large cells and are somewhat larger than the cleaved cells. (See the images below.)
Biopsy of a cervical lymph node showing infiltration with a population of large cells (B cells) consistent with diffuse large cell lymphoma.
Diffuse large B-cell lymphoma. Hematoxylin and eosin stain of a lymph node biopsy sample showing a mixture of large and small cells. The architecture of the node is lost, with a diffuse pattern of involvement.
Pathology of DLBCL
Pathological evaluation is extremely important in the diagnosis of diffuse large B-cell lymphoma (DLBCL). Sufficient biopsy material and formal consultation by an experienced hematopathologist is mandatory. The preferred diagnostic procedure is an excisional biopsy, unless contraindicated because of significant comorbid conditions, the clinical scenario (eg, rapidly growing nodal masses requiring urgent treatment), or the location of nodal/extranodal-involved sites. In cases in which excisional biopsy cannot be performed, multiple core biopsies are acceptable. Fine-needle aspiration (FNA) has a high yield of false-negative results and is not recommended in the workup and diagnosis of patients with a suspected diagnosis of DLBCL or any other forms of lymphoma.
Pathological classification of lymphomas
The classification of lymphomas has undergone significant modifications over the last 3 decades. Currently, 2 classification systems are widely used: the World Health Organization (WHO) and revised European-American (REAL) classification of lymphoid malignancies. [1, 4] The nomenclature of DLBCL has undergone several changes as a result of revisions in the pathological classification of lymphomas over the last decades. It had been previously named a diffuse histiocytic lymphoma (Rappaport’s classification), centroblastic lymphoma (Kiel’s classification), and a large cleaved follicular center cell or large cell immunoblastic lymphoma (working formulation). [1, 4, 3]
Pathological characteristics of DLBCL
Morphologically, DLBCL is composed of large B cells with a high proliferation index resembling germinal centroblasts. DLBCL usually develops de novo but can also emerge as a clonal transformation in patients with low-grade lymphomas or chronic lymphocytic leukemia (CLL). De novo DLBCL tends to have a better response rate to standard therapy and better prognosis than transformed DLBCL. Several morphologic variants of DLBCL have been described, such as centroblastic, immunoblastic, plasmablastic, T-cell/histiocyte-rich, and anaplastic B-cell lymphoma (usually anaplastic lymphoma kinase [ALK]) positive). [49] While each of these variants can be determined by pathological evaluation, the clinical prognostic significance remains controversial.
Immunophenotype studies demonstrate that DLBCL co-expresses pan B-cell markers, including CD19, CD20, CD79a, CD45RA, and the nuclear transcription factor PAX5. The expression of additional markers may have prognostic implications. The proliferation factor Ki67 is usually high, at 65% mean percentage. High Ki67 levels (>80%) have been associated with a shorter survival. [50] Germinal center–associated markers CD10 and Bcl-6 are expressed in approximately 30-40% and 60%, respectively. Bcl-6 expression has been associated with a prolonged progression-free survival (PFS) and overall survival (OS) following rituximab chemotherapy in retrospective studies. [51, 52]
On the other hand, CD5 is expressed only in 10% of DLBCL cases, and its expression should raise the suspicion of transformation from a more indolent form of NHL such as small lymphocytic lymphoma (SLL) or CLL, and it has been associated with a shorter survival. [53]
Subclassification of DLBCL
Under the WHO classification of lymphoid malignancies, the following histological variants are considered clinical and/or pathological distinct subtypes of DLBCL [1] :
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DLBCL not otherwise specified (NOS)
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T-cell/histiocyte-rich large B-cell lymphoma
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Primary DLBCL of the central nervous system (CNS)
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Primary cutaneous DLBCL, leg type
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Epstein-Barr virus–positive (EBV+) DLBCL, NOS
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EBV+ mucocutaneous ulcer
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DLBCL associated with chronic inflammation
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Lymphomatoid granulomatosis
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Primary mediastinal (thymic) large B-cell lymphoma
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Intravascular large B-cell lymphoma
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ALK-positive B-cell lymphoma
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Plasmablastic lymphoma
-
Primary effusion lymphoma
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Human herpesvirus 8 (HHV8)-positive DLBCL
The WHO classification also contains the following entities:
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High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements
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High-grade B-cell lymphoma, NOS
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B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma
Gene expression profiling (GEP) studies provide significant insightful information in the understanding of the DLBCL biology. GEP studies have identified and validated 3 different subtypes of DLBCL, (1) germinal center B-cell (GCB) lymphoma, (1) activated B-cell (ABC) lymphoma, and (3) primary mediastinal lymphoma (PML), each with significant differences in terms of prognosis, PFS, and OS following systemic chemotherapy or, more recently, chemoimmunotherapy. [8, 54, 55]
GCB-DLBCL appears to be derived at the postgerminal state, primarily driven by deregulation of apoptosis by Bcl-6, and has an excellent response to rituximab-based chemoimmunotherapy regimens. ABC-DLBCL is driven by high levels of nuclear factor Kappa-B (NFkB) activity and is associated with a poor outcome, despite chemoimmunotherapy. PML shares GEP signatures similar to those of classical Hodgkin lymphoma and, although it has a good prognosis when compared with other DLBCL subtypes, treatment-related toxicities (ie, involved-field radiation) continue to be a significant problem being addressed in clinical trials.
Although GEP studies are the best way to differentiate different subtypes of DLBCL that might be clinically relevant, and a large prospective trial has demonstrated the feasibility of GEP at diagnosis to subsequently guide therapy, [56] the use of GEP studies is limited by several issues, including cost and availability. [57] Hence, GEP studies are not recommended outside from a clinical trial.
GEP results have been translated into clinically applicable approaches using immunohistochemistry (IHC). [58, 59, 60] IHC appears to be an easy and practical method for differentiating DLBCL subgroups. Several attempts to subtype DLBCL cases into GCB and non-GCB have been made, with algorithms using several markers (eg, CD10, Bcl-6, IRF4/MUM1), such as the Hans algorithm and the algorithm proposed by Muris et al. [58, 59, 61] The Hans algorithm reproduces the gene expression-based classification of DLBCL and has a misclassification rate of 20%. [58] Both algorithms have been evaluated as predictors of clinical outcomes in DLBCL patients undergoing front-line therapy with standard chemotherapy or chemoimmunotherapy. [60, 61] On the other hand, the differences in the clinical behavior and therapeutic response of patients with relapsed/refractory GBC and non-GBC DLBCL have been defined in clinical studies. [62, 13, 14]
Genetic abnormalities in DLBCL
Information obtained from genetic studies performed in DLBCL tumor specimens stresses the complexity in the biology of this disease. DLBCLs express clonally rearranged immunoglobulin H (IgH) genes with somatic mutations in the variable region. For this reason is thought that DLBCL cells are derived from antigen-exposed B-cells. No gene abnormality is pathognomonic for DLBCL. Recurrent translocations involving the BCL6, BCL2, and MYC genes have been described in approximately 50% of cases. Chromosomal translocation leading to up-regulation of BCL2 [t(14;18)] is present in 20-30% of DLBCL cases and is especially observed GCB variants. Gene abnormalities in ABC-DLBCL are more complex and include trisomies, deletions, and chromosomal inactivation. [63]
The clinical value of testing for genetic aberrations in DLBCL continues to grow in recognition. A subset of DLBCL patients carrying both c-Myc and Bcl-2 translocations as detected by fluorescence in situ hybridization (FISH) has been identified. These cases, termed double-hit DLBCL, represent approximately 8% of newly diagnosed DLBCL; they exhibit a poor response to standard doses of rituximab chemotherapy regimens and have a poor OS. [64, 65, 66, 67] Moreover, IHC studies have demonstrated that concurrent over-expression of c-Myc and Bcl-2 is associated with a poor clinical outcome. [68, 69] Currently, c-Myc and Bcl-2 cytogenetic studies (ie, FISH) and IHC analysis for Bcl2 and c-Myc over-expression should be performed in DLBCL patients exhibiting a high proliferation index (ie, Ki67 ≥90%).
Staging
Clinical staging of patients with diffuse large B-cell lymphoma (DLBCL) is fundamental in order to (1) define the treatment plan (ie, combined-modality vs systemic therapy plus/minus CNS prophylaxis), (2) determine risk stratification according to the International Prognostic Index (IPI) score system, and (3) predict the likelihood of survival following frontline therapy.
The staging of lymphoma patients provides information necessary for treatment planning and has prognostic significance, especially in Hodgkin lymphoma (HL). Currently the Ann Arbor staging system is the preferred staging system for DLBCL (see below).
After histologic and immunologic findings confirm the diagnosis of diffuse large cell lymphoma, a pretreatment staging evaluation should be performed. At minimum, patients should have routine blood counts and blood chemistries, particularly a lactate dehydrogenase (LDH) level, which is a prognostic parameter. Carefully examine the peripheral blood smear for any abnormal lymphoid cells.
Note that tumor lysis syndrome manifests as a rapid rise in potassium, phosphorus, and uric acid and a drop in calcium; this can lead to a sudden death from electrolyte abnormalities.
Radiologic staging studies include chest radiography and computed tomography (CT) scanning of the chest, abdomen, and pelvis. Bone, gallium, and PET scans, as well as plain films, may be helpful in selected patients.
Ann Arbor staging system
The Ann Arbor staging system, originally designed for Hodgkin disease, is traditionally used to assess the extent of disease involvement in patients with non-Hodgkin lymphoma (NHL). The stages are characterized as follows:
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Stage I - Involvement of a single lymph node region (I) or localized involvement of a single extralymphatic organ or site (IE)
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Stage II - Involvement of 2 or more lymph node regions on the same side of the diaphragm (II) or localized involvement of a single associated extralymphatic organ or site and its regional lymph nodes, with or without involvement of other lymph node regions on the same side of the diaphragm (IIE)
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Stage III - Involvement of lymph node regions on both sides of the diaphragm (III), which may also be accompanied by localized involvement of an associated extralymphatic organ or site (III E ), by involvement of the spleen (IIIs), or by both (IIIE+S)
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Stage IV - Disseminated (multifocal) involvement of one or more extra lymphatic organs, with or without associated lymph node involvement, or isolated extralymphatic organ involvement with distant (nonregional) nodal involvement
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A - No systemic symptoms present
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B - Unexplained fever ≥ 38º C; drenching night sweats; weight loss ≥ 10% body weight
The Cotswolds modifications are as follows:
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X = bulky disease - Bulky disease is defined as a mass of nodes with one diameter of >10 cm or a mediastinal mass larger than one third of the transthoracic (mediastinal) width
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Number or anatomic regions involved - The number of anatomic regions involved should be indicated by a subscript (eg, II3)
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Biopsy of a cervical lymph node showing infiltration with a population of large cells (B cells) consistent with diffuse large cell lymphoma.
-
Computed tomography (CT) scan of the abdomen showing mesenteric and retroperitoneal adenopathy in a patient with diffuse large cell lymphoma.
-
Diffuse large B-cell lymphoma. Hematoxylin and eosin stain of a lymph node biopsy sample showing a mixture of large and small cells. The architecture of the node is lost, with a diffuse pattern of involvement.
-
Patient with diffuse large B-cell lymphoma with extranodal involvement. This computed tomography (CT) scan shows an enlarged spleen and liver as a result of lymphomatous involvement.
-
Patient with diffuse large B-cell lymphoma with extranodal involvement (same patient as in the previous image). This patient has an enlarged spleen and liver as a result of lymphomatous involvement. Extensive retroperitoneal lymphadenopathy is also present.
-
This image depicts gallium scans performed as part of a staging workup for a patient with diffuse large B-cell lymphoma. The scans show extensive hepatosplenic and multiple sites of nodal involvement with a gallium-avid tumor.
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Role of B-cell receptor signaling in promoting proliferation and survival of DLBCL. BCR is the main factor in B-cell biology, playing a key role in B-cell development, antigen-driven clonal selection, and humoral immunity. B-cell receptor signaling activates PI3K-mediated activation of the kinase AKT, which activates many downstream signaling pathways. All these downstream pathways are essential for the survival of B cells. PIP3 is generated as a result of BCR-dependent PI3K activation. BTK also hydrolyzes PIP2 into DAG and IP3. IP3 induces release of calcium stores from the endoplasmic reticulum. Ca and DAG activate PKC, which leads to activation of NF-k B pathyway. PI3K, phosphatidylinositide-3 kinase; AKT, protein kinase B; PTEN, phosphatase and tensin homolog; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-4,5-trisphosphate; IKK, IkB kinase; mTOR, mammalian target of rapamycin; FoxO, Forkhead box transcription factors; GSK3b, glycogen synthase 3-beta; p21, inhibitor of cyclin-dependent kinases.
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