The World Health Organization (WHO) publishes the definitive classification system that pathologists worldwide use to diagnose and categorize lymphoma. Now in its 5th edition, the WHO Classification of Haematolymphoid Tumours organizes dozens of lymphoma subtypes based on a combination of tissue appearance under the microscope, protein markers on cell surfaces, genetic abnormalities, and clinical features. A lymphoma diagnosis isn’t a single test. It’s a layered process that moves from biopsy to microscope to molecular analysis, each step narrowing down exactly which type of lymphoma is present.
How a Lymphoma Diagnosis Starts
The process begins with a biopsy of an enlarged lymph node or affected tissue. The gold standard is a surgical excisional biopsy, where an entire lymph node is removed and sent to a pathologist. This matters because lymphoma diagnosis depends on seeing the overall architecture of the tissue, not just individual cells. A large French study comparing biopsy methods found that surgical excisions provided a definitive diagnosis in 98.1% of cases, compared to 92.3% for core needle biopsies. Core needle biopsies were also 18 times more likely to yield insufficient material (1.8% vs. 0.1%).
Fine-needle aspiration, the thin-needle technique commonly used for other conditions, is generally not adequate for lymphoma. It can’t capture enough tissue structure to distinguish between subtypes, and it can’t reliably identify the hallmark cells needed for certain diagnoses, such as the Reed-Sternberg cells required for Hodgkin lymphoma.
What the Pathologist Looks For
Once tissue is obtained, the pathologist examines it in layers. The first step is morphology: the size, shape, and arrangement of cells under the microscope. Lymphoma cells may be large or small, grow in sheets or clusters, and disrupt the normal architecture of the lymph node in characteristic ways. In classic Hodgkin lymphoma, for example, the actual cancer cells are relatively rare, scattered among a background of normal inflammatory cells. These malignant cells, called Reed-Sternberg cells, are distinctively large with prominent nucleoli and sometimes multiple nuclei.
Diffuse large B-cell lymphoma (DLBCL), the most common aggressive lymphoma, shows large abnormal cells that have overtaken the normal lymph node structure. The WHO recognizes three morphological subtypes: centroblastic, immunoblastic, and anaplastic, each with a different cellular appearance.
Protein Markers That Identify Cell Type
Morphology alone can’t distinguish most lymphoma subtypes. The next critical step is immunohistochemistry, a technique that uses antibodies to detect specific proteins on the surface of tumor cells. These proteins act like identity badges, revealing whether the lymphoma arises from B cells, T cells, or NK cells, and often pointing to a specific subtype.
Two markers form the foundation of nearly every lymphoma workup:
- CD20: a protein found on the surface of most mature B-cell lymphomas
- CD3: a protein found on most mature T-cell lymphomas
From there, pathologists apply panels of additional markers tailored to what the morphology suggests. For Hodgkin lymphoma, the key combination is CD30 (strongly positive on Reed-Sternberg cells) and CD15, while CD20 is typically negative. A variant called nodular lymphocyte-predominant Hodgkin lymphoma flips this pattern: tumor cells are CD20-positive and CD30-negative, which is one reason accurate marker testing is essential.
For DLBCL, pathologists check a panel that includes Ki-67 (a measure of how fast cells are dividing, typically above 80% in DLBCL), along with markers like CD10, BCL-6, and MUM-1. These three markers feed into an algorithm that classifies the lymphoma as either germinal center B-cell type or activated B-cell type, a distinction that carries prognostic significance.
Chronic lymphocytic leukemia and small lymphocytic lymphoma share a characteristic fingerprint: tumor cells that are CD20-positive with co-expression of both CD5 and CD23. Mantle cell lymphoma also expresses CD5 but lacks CD23, and its hallmark is overexpression of a protein called Cyclin D1.
T-Cell and NK-Cell Lymphomas
T-cell lymphomas require their own specialized panels. Pathologists look for “pan-T-cell” markers (CD2, CD3, CD5, CD7) and then assess whether cells are CD4-positive or CD8-positive, which helps narrow the subtype. A key diagnostic clue is the loss or abnormally dim expression of one or more of these expected T-cell markers, which suggests a malignant rather than reactive process.
One important exception: anaplastic large cell lymphoma is technically a T-cell lymphoma, but its cells are usually CD3-negative. Instead, they show strong, uniform CD30 positivity. Diagnosing the ALK-positive subtype requires demonstrating the presence of the ALK protein, which also influences treatment decisions and prognosis. For follicular helper T-cell lymphomas, pathologists look for at least two or three markers from a specific set (PD-1, ICOS, CXCL13, CD10, BCL-6) expressed on CD4-positive cells.
NK cells and T cells can be distinguished because NK cells express a form of CD3 detectable inside the cell but not on its surface, and they lack CD5. This distinction matters for entities like extranodal NK/T-cell lymphoma, where confirming Epstein-Barr virus infection in the tumor cells through a technique called in situ hybridization is part of the diagnostic criteria.
Genetic Testing for Subtype Classification
Many lymphoma subtypes are defined, at least in part, by specific chromosomal rearrangements. These are breaks in chromosomes that move genes next to each other in ways that drive cancer growth. Identifying these rearrangements has moved from optional to essential for accurate classification under the current WHO system.
Follicular lymphoma, the most common slow-growing lymphoma, carries a translocation between chromosomes 14 and 18 in about 85% of cases. This rearrangement places the BCL2 gene (which prevents cell death) under the control of an antibody gene’s promoter, causing the protein to be overproduced.
Burkitt lymphoma is defined by rearrangement of the MYC gene, most often a translocation to chromosome 14 (about 80% of cases), with chromosomes 2 and 22 accounting for the rest. Notably, Burkitt lymphoma does not carry BCL2 or BCL6 rearrangements. If those are present alongside MYC, the diagnosis shifts to a different category.
That category, high-grade B-cell lymphoma, specifically requires the identification of “double-hit” genetics: MYC rearrangement combined with either BCL2 or BCL6 rearrangement. These lymphomas behave more aggressively than standard DLBCL, and their recognition as a distinct entity in the WHO classification changed how they’re treated. For any new DLBCL diagnosis, testing for MYC, BCL2, and BCL6 rearrangements is now considered a minimum standard.
Mantle cell lymphoma is defined by the translocation between chromosomes 11 and 14, which drives overexpression of Cyclin D1 in roughly 95% of cases. Marginal zone lymphomas of the stomach and other extranodal sites carry their own set of translocations, with the most common being t(11;18), a rearrangement specific to extranodal marginal zone lymphoma that also predicts a lower chance of responding to antibiotic therapy in stomach cases.
How the WHO Classifies Subtypes
The 5th edition of the WHO classification, published in 2022, reorganized lymphomas into a hierarchical system. Major categories separate B-cell neoplasms from T-cell and NK-cell neoplasms, with further divisions based on whether cells are immature (precursor) or mature. Within each branch, individual entities are defined by the combination of features described above: morphology, protein markers, genetic findings, and clinical behavior.
Several notable changes arrived with this edition. Follicular lymphoma grading (the old 1 through 3A scale) is no longer mandatory. The previous grade 3B category has been renamed follicular large B-cell lymphoma for consistency. Distinct variants like pediatric-type follicular lymphoma, duodenal-type follicular lymphoma, and in-situ follicular B-cell neoplasm are now recognized as separate entities rather than subcategories. The classification also added germline predisposition syndromes, acknowledging that some people carry inherited genetic variants that increase lymphoma risk.
For DLBCL, the cell-of-origin classification into germinal center B-cell and activated B-cell subtypes remains central. While gene expression profiling is the most accurate method, the WHO continues to accept immunohistochemistry-based algorithms as a practical substitute, since gene expression testing isn’t available everywhere.
Staging After Diagnosis
Once a lymphoma subtype is confirmed, the next step is determining how far it has spread. The Lugano classification, adopted internationally, defines four stages:
- Stage I: involvement of a single lymph node region or a single organ site outside the lymphatic system
- Stage II: two or more lymph node regions on the same side of the diaphragm (the muscle separating the chest from the abdomen)
- Stage III: lymph node regions on both sides of the diaphragm, or nodes above the diaphragm with spleen involvement
- Stage IV: widespread involvement of organs outside the lymphatic system, including the bone marrow, liver, lungs, or cerebrospinal fluid
PET/CT scanning is the primary imaging tool for staging most lymphomas, particularly Hodgkin lymphoma and aggressive B-cell lymphomas, because it can detect metabolically active disease throughout the body in a single scan. Stage II disease can also be designated “bulky” when a mass exceeds a certain size threshold, which influences treatment planning.