Practical Examples in Lymphoma Pathology

Role and Limitations of Fine Needle Aspiration (FNA) in Lymphoma

• Fine needle aspiration biopsy (FNA) involves inserting a needle, typically the size of a local anesthetic needle, into a mass to aspirate a small number of cells for examination.

• FNA is highly effective for identifying many types of cancers, such as metastatic carcinoma or melanoma, and is often the only biopsy required for those diagnoses.

• FNA is a useful screening tool for clinicians to differentiate between metastatic cancer, lymphoma, or infection in an outpatient setting or radiology suite.

• Despite its utility for other cancers, FNA is insufficient for the definitive first-time classification of lymphoma for the following reasons:

  • Need for Architecture: A definitive lymphoma diagnosis requires viewing the overall architecture of the lymph node tissue, which is destroyed or not captured in a cell-only aspirate.

  • Subclassification Requirements: Subclassifying non-Hodgkin lymphoma is necessary to guide specific treatments. A pathologist cannot meaningfully subclassify lymphoma purely on FNA and may only report findings as "atypical lymphocyte presence."

  • Diagnostic Certainty: FNA cannot always differentiate between a malignant process and reactive conditions like glandular fever (infectious mononucleosis).

• Pathologists may provide an "off-the-record" suspicion of lymphoma based on FNA, but a core biopsy or a whole node excision is required for formal diagnosis.

• FNA may be sufficient in cases where a patient has a known history of lymphoma and the clinician simply needs to confirm if a new lump is a recurrence or an infection.

Case Study: Chronic Lymphocytic Leukemia/Lymphoma (CLL/SLL)

• Patient Presentation: A 75-year-old male with long-standing lymphadenopathy.

• Macroscopic Findings:

  • The excised lymph node is enlarged, measuring approximately $5\,cm$.

  • The cut surface has a pale, uniform appearance described as "fish flesh," resembling the cut surface of a barramundi fish.

  • This contrasts with normal lymph nodes, which are typically darker in color.

• Microscopic Findings:

  • At low power, the architecture is clearly abnormal; the normal germinal centers are absent.

  • At high power, the tissue shows a sheet-like growth pattern of small lymphocytes.

  • While individual cells might appear benign in isolation, their monotony and sheet-like growth in the biopsy confirm malignancy.

• Immunohistochemistry (IHC) and Phenotype:

  • The cells are positive for $CD20$ (a B-cell marker).

  • In this specific case, the cells were also positive for $CD5$ and $CD23$.

• Clinical Context: This phenotype is typical for Chronic Lymphocytic Lymphoma (CLL). Because many patients also have circulating small lymphocytes in their blood, the condition is often referred to as Chronic Lymphocytic Lymphoma/Leukemia.

Mimics of Lymphoma and the Importance of Viral Serology

• Certain viral infections can clinically and microscopically mimic lymphoma, leading to potential diagnostic errors.

• Acute Epstein-Barr Virus (EBV): Infections like acute glandular fever can look remarkably like lymphoma under the microscope.

• Screening Protocol: Practically all specialists perform a comprehensive viral serology panel before proceeding to a biopsy for suspected lymphoma. This includes testing for:

  • Epstein-Barr Virus (EBV)

  • Cytomegalovirus (CMV)

  • Human Immunodeficiency Virus (HIV)

  • Hepatitis C

• Pathologists prefer to know that viral serology is negative before examining a lymph node to reduce the risk of a false-positive diagnosis of malignancy.

Case Study: Burkitt Lymphoma and Diagnostic Techniques

• Patient Presentation: A 23-year-old male with an enlarged neck node and negative viral serology.

• Morphology:

  • The node exhibits the "fish flesh" appearance.

  • Microscopic examination at low power reveals white areas scattered within a blue cellular background, known as a "starry sky" appearance (reminiscent of Vincent van Gogh's painting The Starry Night).

  • The cells have characteristic nuclei associated with Burkitt lymphoma.

• Flow Cytometry Analysis:

  • Forward and Side Scatter: Indicates the size and complexity of the lymphocytes.

  • Phenotypic Markers: $95\%$ of cells are $CD19$ positive and $CD5$ negative. They are also $CD10$ positive and $CD22$ negative. Some reveal a $CD20$ positive and $CD2$ negative phenotype.

  • Light Chain Restriction: In chronic inflammation, the ratio of lambda ($\lambda$) to kappa ($\kappa$) light chains should be roughly equal ($50/50$). In this case, $96\%$ of the cells expressed the lambda light chain and very few expressed kappa. This "light chain restriction" is definitive evidence of a clonal (monoclonal) population.

• Fluorescence In Situ Hybridization (FISH):

  • Burkitt lymphoma is defined by a specific genetic translocation, typically involving the $MYC$ gene.

  • FISH uses DNA and RNA probes (e.g., a red probe for $MYC$ on Chromosome $8$ and a green probe for $IGH$ on Chromosome $14$).

  • In normal cells, these chromosomes are far apart. In this patient, the red and green probes were located immediately adjacent to each other, confirming a translocation between Chromosome $8$ and $14$, represented as $t(8;14)$.

  • This confirmatory genetic test is critical because Burkitt lymphoma requires a distinct treatment regimen and has a unique natural history.

Case Study: Follicular Center Cell Lymphoma

• Patient Presentation: A 50-year-old with an enlarged groin lymph node.

• Microscopic Architecture:

  • The architecture initially appears to have follicles, but there are too many of them, and they lack orderly arrangement.

  • The follicles lack "tingible body macrophages," which are usually present in normal follicles to clean up debris.

  • The mantle and marginal zones are not well-polarized, making the follicles appear as a distorted parody of normal tissue.

• Immunohistochemistry:

  • The cells are $CD20$ positive (B-cell marker).

  • The follicles express $BCL2$. Critically, normal benign follicles do not express the $BCL2$ protein. This expression serves as a primary diagnostic clue for follicular center cell lymphoma.

• Ancillary Testing:

  • Flow Cytometry: Shows a kappa ($\kappa$) light chain restricted population that is $CD38$ positive.

  • FISH: Can be used to show the $t(14;18)$ translocation characteristic of this lymphoma, though it is not always required if morphology and IHC are definitive.

• Grading: These lymphomas are graded (e.g., grade $2$ or $3$) to determine the appropriate treatment pathway.

Case Study: Reactive Mimicry in the Thyroid

• Patient Presentation: A 65-year-old with a thyroid mass, initially suspected to be MALT lymphoma based on preoperative FNA and flow cytometry.

• Pathological Review:

  • Excision showed follicles and thyroid epithelium with lymphocytes in the background, but no sheet-like growth of B-cells ($CD20$).

  • Clonality Testing: IHC for kappa and lambda light chains showed an equal distribution, indicating a polyclonal (reactive) population rather than a neoplasm.

  • Molecular Testing: Next-generation sequencing (NGS) was used to sequence thousands of lymphocytes. If it were lymphoma, a clonal B-cell receptor gene rearrangement or specific DNA translocation would be present. No such markers were found.

• Diagnosis: The condition was confirmed as chronic Hashimoto's thyroiditis, a completely benign inflammatory condition, preventing unnecessary lymphoma treatment.

Case Study: Misdiagnosis and T-cell Rich B-cell Lymphoma

• Patient Presentation: A 35-year-old male previously diagnosed with Hodgkin lymphoma five years prior, now presenting with a relapse after radiation treatment.

• Diagnostic Skepticism: Most Hodgkin lymphoma cases have an $80\text{--}90\%$ cure rate. A relapse suggests the original diagnosis might be incorrect.

• Microscopic Discovery:

  • The excision showed a nodular and sclerosing appearance with large cells resembling Reed-Sternberg (RS) cells (multilobated, "owl-eye" nuclei).

  • IHC for $CD3$ highlighted benign T-cells in the background.

  • IHC for $CD30$ (typically positive in Hodgkin lymphoma) was negative.

  • IHC for $CD20$ was positive in the large RS-like cells.

• Re-classification: Classic Hodgkin lymphoma cells do not usually express $CD20$. These cells were actually B-cells.

• The Correct Diagnosis: The original slides were retrieved from archives, revealing the patient always had T-cell rich B-cell lymphoma (a subtype of diffuse large B-cell lymphoma).

• Clinical Impact: This subtype has a worse prognosis and requires different treatment than Hodgkin lymphoma. The patient eventually achieved remission with appropriate salvage treatment.

Core Principles of Lymphoma Pathology

• Hodgkin vs. Non-Hodgkin: The primary distinction is the presence of Reed-Sternberg cells, which must not only look correct morphologically but also express specific proteins (e.g., usually $CD30$ positive, negative for most B-cell markers).

• Tissue Requirements:

  • Architecture: Vital for classification.

  • Fresh Tissue: Required for flow cytometry, which is essential for determining clonality via light chain restriction.

  • Communication: Clinicians must notify the lab when sending fresh tissue to ensure it is processed before the pathology staff leaves and before the tissue decomposes.

• Classification Complexity: Non-Hodgkin lymphoma includes many different tumors with varying natural histories and treatments. Subclassification should only be performed by experienced pathologists with subspecialty expertise using the World Health Organization (WHO) endorsed classification system.