Advanced Study Notes on non-Hodgkin Lymphoma and WHO Classification and Pathological Practical Considerations

Overview of Lymphoma Classifications

• Lymphomas are broadly divided into two categories: Hodgkin and non-Hodgkin lymphoma.

• Approximately $30\%$ of lymphomas are categorized as Hodgkin lymphoma, while the remaining $70\%$ are non-Hodgkin lymphoma.

• While Hodgkin lymphoma represents a fairly uniform group with consistent behavior despite varied microscopic appearances, non-Hodgkin lymphoma (NHL) is a highly diverse group of different tumors.

The Importance of Subtyping non-Hodgkin Lymphoma

• NHL tumors are malignant tumors of lymphocytes. Their behavior ranges from extremely low-grade (indolent) to extremely high-grade (aggressive).

• Low-grade lymphomas may persist for years and often require only observation.

• High-grade lymphomas can be fatal within weeks or months if left untreated.

• Accurate subtyping is critical because NHL encompasses many different diseases with distinct natural histories, treatments, and outcomes. A diagnosis of "non-Hodgkin lymphoma" alone is insufficient for clinical management.

Evolution of Classification Systems

• Several historical classification systems exist, but many are now redundant and should be ignored if found in textbooks:

  • Rappaport Classification ($1970$s): Used terms like "well differentiated lymphocytic lymphoma" and "poorly differentiated." It is historical and no longer in use.

  • Kiel Classification ($1974$): Developed in Kiel, Germany; it made progress by classifying tumors by both morphology and protein expression. It is now redundant.

  • International Working Formulation ($1982$): Divided lymphomas into low, intermediate, and high grade. It was common until $2000$ but is now redundant.

  • Revised European American Lymphoma (REAL) Classification ($1994$): A major advancement that classified lymphomas based on cell of origin, morphology, immunology, genetics, and clinical features.

  • World Health Organization (WHO) Classifications: This is the current global consensus standard.

    • WHO $2001$: The third edition and the first to establish a universal system.

    • WHO $2017$ ($4\text{R}$): The fourth edition revised, which is the current standard for planning treatment.

    • WHO $2022/2023$: The fifth edition, often referred to as the "Blue Book," which is increasingly available online via subscription.

The WHO Consensus Process

• The WHO classification is determined at meetings in Lyon, France.

• The WHO is part of the United Nations; the meeting site is considered UN soil rather than French soil.

• Experts from around the world gather to set arbitrary but agreed-upon rules for diagnosis based on morphology and protein expression.

• Complex diagnostic difficulties are often resolved through consensus during these sessions, sometimes aided by the local Leonese cuisine.

Cellular Markers and Immunophenotyping

• Lymphocytes (B cells, T cells, and NK cells) cannot be reliably distinguished by morphology alone under a microscope.

• Classification relies on the expression of specific proteins, often designated as Cluster of Differentiation (CD) markers.

  • CD3: A primary marker for T cells and T cell tumors.

  • CD20: A primary marker for B cells and most B cell lymphomas.

  • CD4 and CD8: Markers for helper and killer T cells, respectively.

  • CD5: Normally a T cell marker, but it is aberrantly expressed in certain B cell lymphomas (e.g., Chronic Lymphocytic Leukemia/Lymphoma).

  • CD10: Expressed by benign lymphocytes in the germinal follicles and is a defining feature of follicular center cell type lymphoma.

  • CD19: A common B cell marker.

  • CD23: Often positive in Chronic Lymphocytic Leukemia (CLL).

  • Cyclin D1: A marker used to identify mantle cell lymphoma.

Diagnostic Techniques

• Immunohistochemistry (IHC):

  • Involves applying antibodies (e.g., anti-CD20) to tissue on a glass slide.

  • A secondary agent causes cells expressing the target protein to turn brown, allowing for identification under a microscope.

• Flow Cytometry:

  • Requires fresh tissue or blood to create a cell suspension.

  • Cells pass through a laser which measures size and shape through "side scattering" and "forward scattering."

  • Antibodies with specific charges are attached to the cells; magnets then deflect the cells into different categories based on their expressed markers.

  • Results are typically displayed on an X-Y axis plot showing populations that are positive or negative for specific markers (e.g., a population positive for both CD19 and CD10).

  • Fresh tissue is mandatory for this technique as formalin fixation prevents its use.

Lymph Node Architecture and Lymphoma Origin

• The classification of B cell lymphomas often correlates with the specific region of the lymph node from which the tumor arises:

  • Germinal Center: Contains follicular center cells. Markers: CD10 positive ($+$), CD5 negative ($-$), Cyclin D1 negative ($-$). Tumors here are Follicular Center Cell Lymphomas.

  • Mantle Zone: Surrounds the germinal center. Markers: CD5 positive ($+$), Cyclin D1 positive ($+$), CD10 negative ($-$). Tumors here are Mantle Cell Lymphomas.

  • Marginal Zone: Outer region. Markers: CD5 negative ($-$), CD10 negative ($-$), Cyclin D1 negative ($-$). Tumors here are Marginal Zone Lymphomas.

  • Mucosal Associated Lymphoid Tissue (MALT): Often found in the gastrointestinal tract (GIT). These are also called extranodal marginal zone lymphomas.

Genetics and Cytogenetics

• Modern diagnosis utilizes genetics and fluorescence in situ hybridization (FISH) rather than older, slower karyotyping methods that required growing cells in a lab for weeks.

• Specific lymphomas are defined by recurrent translocations:

  • Burkitt Lymphoma: Characterized by the $t(8;14)$ translocation.

  • Anaplastic Large Cell Lymphoma: Characterized by the $t(2;5)$ translocation, leading to the expression of the $ALK$ gene.

Clinical Profiles of non-Hodgkin Lymphoma Subtypes

• Diffuse Large B-Cell Lymphoma (DLBCL):

  • Accounts for more than one-third ($> 33.3\%$) of all NHL cases.

  • Aggressive but highly curable.

  • Five-year survival rate is approximately $70\%$ to $80\%$ with R-CHOP chemotherapy.

• Burkitt Lymphoma:

  • Comprises $2.5\%$ of lymphomas.

  • Extremely rapid growth but more than $90\%$ are cured with specific, highly intensive chemotherapy.

• Anaplastic Large Cell Lymphoma:

  • Comprises $1.8\%$ of cases.

  • Aggressive but has a good survival rate; can be treated with targeted therapy against the expressed $ALK$ gene.

• MALT Lymphoma:

  • Indolent and sensitive to radiotherapy.

  • In the stomach, it can sometimes be treated solely by eradicating Helicobacter pylori.

• Mantle Cell Lymphoma:

  • Comprises $2.4\%$ of cases.

  • Median survival of only $3$ to $5$ years.

  • Indolent growth but inherently progressive and generally considered incurable without a bone marrow transplant.

• Chronic Lymphocytic Leukemia/Lymphoma (CLL):

  • Low-grade and indolent; five-year survival is $51\%$ with a median survival of $7$ years.

  • Primary clinical issues often stem from immunosuppression rather than tumor mass (e.g., high vulnerability to COVID-19).

Transformation of Lymphomas

• Transformation occurs when a low-grade lymphoma acquires additional mutations and becomes high-grade and aggressive.

  • Richter’s Transformation: The specific term for when CLL transforms into Diffuse Large B-cell Lymphoma.

  • Follicular Transformation: Follicular center cell lymphomas can progress from Grade 1 to 2 to 3, eventually becoming DLBCL.

  • MALT Transformation: Extranodal marginal cell lymphomas can also acquire mutations to transform into DLBCL.

Practical Clinical and Laboratory Considerations

• Diagnosis Limitations: Fine Needle Aspiration (FNA) should not be used to diagnose lymphoma because it does not allow for the evaluation of tissue architecture or the performance of comprehensive flow cytometry.

• Tissue Handling: Fresh tissue is essential for flow cytometry. Clinicians must communicate with the pathology lab before sending samples to ensure staff are available to process the tissue before it decomposes (e.g., avoiding samples arriving on Friday afternoons when they might sit over a weekend).