Diagnosing Hematolymphoid Neoplasms

A brief forward: Many thanks to Drs. Corliss Newman, Alireza Torabi, and Sindhu Cherian for contributing this material.

OVERVIEW:

Understanding hematopoietic neoplasms requires knowledge of how their classification has evolved over time. Initially, these neoplasms were categorized based on clinical presentation and cell morphology—how the cells appeared under the microscope and the symptoms patients presented with (e.g., adenopathy, elevated WBC count). Malignant cell morphology often reflects mutations that disrupt normal precursor cell maturation, and early naming conventions were based on these observations. This is why a solid understanding of hematopoiesis is essential. For example, in prolymphocytic leukemia, high WBC counts correspond to an excess of immature promyelocytes, while in chronic lymphocytic leukemia (CLL), the blood is filled with more mature lymphocytes.

As technology has progressed and our understanding of cell biology has expanded, the classification of hematologic neoplasms evolved to include both flow cytometry and evaluation for genetic mutations. Flow cytometry identifies specific cell surface markers, while genetic alterations, such as mutations and translocations, play a critical role in classifying many lymphomas and leukemias, alongside morphology and immunophenotyping.

These advances have significantly shaped the development of treatments for hematologic malignancies. Initially, chemotherapy was the primary treatment approach, targeting rapidly dividing cancer cells. However, with a deeper understanding of cell surface markers and genetics, more targeted therapies have been developed. For example, rituximab, a monoclonal antibody, specifically targets the CD20 cell surface marker on B-cells. As you study hematologic malignancies, keep in mind these foundational concepts—they will aid your understanding of how modern diagnostic tools and treatments have evolved.

Leukemia vs. Lymphoma:

Historically, “leukemia” referred to malignancies with elevated peripheral white blood cell (WBC) counts, and subtypes were defined by cell morphology. Similarly, “lymphoma” was used for lymphoid cancers originating in lymph nodes, with subtypes determined by the appearance of cancerous lymphocytes. Interestingly, there is significant overlap between some leukemias and lymphomas. For example, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are essentially the same disease. In CLL, cancerous lymphocytes are found primarily in the blood, while in SLL, they are concentrated in lymph nodes. Treatment of both is identical.

White Blood Cell Neoplasms, which arise from hematopoietic stem cell (HSCs), can be divided into two classes:

• Lymphoid Neoplasms include lymphomas, lymphoid leukemias, and plasma cell neoplasms. These cancers originate from early lymphoid progenitor cells and are driven by mutations that affect maturation or growth factor independence. Lymphoid malignancies can have a blood or a lymph node predominant form.
• Myeloid Neoplasms arise from early myeloid progenitors with similar mutation-driven disruptions in maturation or growth regulation. Myeloid malignancies tend to mainly involve blood, although myeloid malignant cells can migrate to other tissues/involve other tissues.

Diagnosing hematolymphoid neoplasms involves several key components:

1. Clinical History and Physical Examination

• • Lymphoid Neoplasms: Symptoms often include lymphadenopathy, mediastinal mass, splenomegaly, or extranodal masses (e.g., skin, brain). “B symptoms” (fever, night sweats, weight loss) and signs of bone marrow involvement (weakness, fatigue, infections, bleeding) are also common. Plasma cell neoplasms can cause bone lesions/pathologic fracture while secretion of Ig fragments or antibodies can result in autoimmune cytopenia or organ damage.
  • Myeloid Neoplasms: Common symptoms include fatigue, weakness, fever, infections, and abnormal bleeding, while a physical exam may demonstrate pale conjunctiva, splenomegaly, hepatomegaly, a high platelet count, or bruising (ecchymoses, purpura, or petechiae). A complete blood count (CBC) may reveal cytosis (e.g. reactive, AML, or MPN) or cytopenia (e.g., due to infection, nutritional, drug effects, AML, or MDS).

2. Tissue Acquisition

• • Peripheral Blood Smears help assess the morphology of blood cells, identify abnormalities, and guide further testing.
    • After whole blood is centrifuged, its components separate into distinct layers. The buffy coat forms a thin, whitish layer between the red blood cells and plasma, consisting primarily of white blood cells and platelets. Observing this layer provides a concentrated view of leukocytes, offering valuable insights into various hematologic conditions.

• • Bone Marrow Biopsy and Aspirate: Uses a Jamshidi needle at the posterior iliac crest to evaluate hematologic neoplasms, stage lymphoma, assess marrow failure, and monitor therapy response.
  • Lymph Node Biopsy: Excisional or core needle biopsy for evaluating hematologic neoplasms in cases of persistent lymphadenopathy or masses.

3. Histologic evaluation

• • Bone marrow biopsies help assess cellularity, differentiation, and blast count. Hypercellularity is seen in conditions like acute leukemia and myeloproliferative neoplasms, while hypocellularity may indicate aplastic anemia or hypoplastic MDS
  • In a lymph node biopsy, the main focus is on evaluating the architecture and identifying any abnormal cell populations, such as clusters of atypical lymphocytes or disrupted germinal centers, which can indicate lymphoma.

4. Immunophenotyping

• • Immunohistochemistry: Performed on formalin-fixed, paraffin-embedded tissue to detect specific proteins.
  • Flow Cytometry: A rapid and sensitive, semi-quantitative technique that uses fresh tissue to analyze cell populations based on surface markers.

5. Genetic Testing

• • Karyotyping: Detects numeric and structural chromosomal abnormalities by culturing dividing cells and examining metaphase spreads.
  • Fluorescent In Situ Hybridization (FISH): Identifies specific chromosomal abnormalities using labeled probes, offering faster results than karyotyping.
  • Molecular Methods: Techniques such as PCR and high-throughput next generating sequencing allow for detailed genetic analysis, including the detection of oncogenic mutations and clonality.

BONE MARROW CELLULARITY AND EXAMINATION:

• Bone marrow cellularity varies by age, with younger individuals having more hematopoietic cells compared to fat cells
  • Normal bone marrow cellularity= 100%-patient age
  • Hypercellular bone marrow can indicate acute leukemia, myeloproliferative neoplasms, myelodysplasia, or involvement by plasma cell neoplasms/ lymphoma/ carcinoma. May also be seen in reactive cases such as nutritional deficiency
  • Hypocellular bone marrow is seen in conditions like aplastic anemia, hypoplastic MDS, paroxysmal nocturnal hemoglobinuria, genetic disorders (such as Fanconi anemia), fibrotic bone marrow, or after certain treatments.

• Bone marrow aspirates:

LYMPH NODE ARCHITECTURE:

• Understanding the architecture of a normal lymph node is essential when evaluating lymphadenopathy and identifying abnormal structures like expanded germinal centers or malignant cell infiltration. Recall from your time in I&I, a normal lymph node consists of several distinct regions:
  • Cortex: Contains B-cell follicles, where B cells undergo proliferation and differentiation.
  • Paracortex: The T-cell-rich zone.
  • Medulla: Contains plasma cells and histiocytes.

IMMUNOPHENOTYPING:

Immunophenotyping (including flow cytometry) is crucial for distinguishing between hematolymphoid malignancies, using specific markers to identify cell lineage and maturity:

• Leukemia:
  • Markers of Immaturity: CD34, TdT
  • Myeloid Markers: CD117, CD13, CD15, CD33, myeloperoxidase (MPO)
• Lymphoma:
  • B-cell Markers: CD19, CD20, kappa, lambda
  • T-cell Markers: CD2, CD3, CD4, CD5, CD7, CD8

Immunohistochemistry is a technique used to detect specific antigens in tissue sections by using antibodies tagged with a chromogen, allowing for visualization of the location and abundance of proteins to help diagnose and classify diseases, particularly cancers.

Flow cytometry allows for the detailed characterization of cellular populations based on their size, granularity, and antigen expression (immunophenotype).

• Sample type: Flow cytometry requires fresh samples, ideally processed within 24 hours. Samples that can be used include liquid samples (peripheral blood, bone marrow aspirates, and body fluids such as cerebrospinal fluid and ascitic fluid) and solid tissues (lymph node biopsies and other tissue samples after they are disaggregated into a cell suspension).
• How flow cytometry works: Flow cytometry involves suspending cells in a fluid stream and passing them through a laser beam. As the cells pass through, the following properties are measured:
  • Forward Scatter (FSC): Reflects cell size.
  • Side Scatter (SSC): Reflects cytoplasmic complexity or granularity.

• • Additionally, flow cytometry detects the presence of specific antigens on the surface or within the cells by using fluorescently labeled antibodies. The level of fluorescence emitted by the labeled cells correlates with the density of antigen expression, allowing for precise immunophenotyping.

• Immunophenotyping in hematologic neoplasms: For example, the sample below is stained with distinct color markers (e.g., orange, blue, green), which are evaluated alongside forward and side scatter to precisely classify the neoplasm.

• As many samples are complex and contain different cell types (as seen in the bone marrow aspirate on the left below), analysis starts by identifying a population of interest (Note: CD45 versus SSC analysis is a good first step). Once the population of interest is identified, a gate is drawn around it (to the right) so that antigen expression might be specifically evaluated in that population. If there is concern for leukemia, blasts are gated; if there is concern of lymphoma, lymphocytes are gated.

• Examples:
  • AML vs ALL

• • B-cell neoplasms

GENETIC AND MOLECULAR DIAGNOSTICS:

Genetic abnormalities play a significant role in the pathogenesis of many hematolymphoid neoplasms:

• Chromosomal Translocations: Certain lymphomas, like Burkitt lymphoma, are characterized by translocations: e.g., t(8;14) involving c-myc
  • Below is a normal karyotype: karyotypes require dividing cells (culture 24-48 hours) and review of metaphase spread. While they can identify numeric and structural abnormalities, they have low resolution and sensitivity.

• • And this is an example of karyotype in CML, which demonstrates the classic translocation t(9;22).

• • And here is an example of Fluorescent in situ hybridization (FISH). While FISH is faster than conventional karyotyping, one has to know exactly what they are looking for (i.e. Probes are gene specific)

• Clonality studies are used to determine whether a population of cells, particularly lymphocytes, arises from a single clone, which helps distinguish between reactive (polyclonal) processes and malignant (monoclonal) neoplasms, aiding in the diagnosis of lymphoid malignancies.

• Molecular Sequencing: High-throughput sequencing allows for the identification of mutations in a broad range of genes, facilitating a more precise diagnosis and prognosis.