Diagnostics for lymphoma: cytologic morphology, flow phenotype and receptor rearrangement (Proceedings)

Lymphocyte types

Small lymphocytes are smaller in size than a neutrophil and have a round nuclei that takes up the majority of the cell.  The nuclei contain densely aggregated chromatin forming large chromocenters (condensed chromatin).  Nucleoli are not seen.  The cytoplasm is scant (sometimes only a very thin rim is visible) and lightly basophilic in color.  These are typically called ‘mature lymphocytes'.  However, early lymphoid progenitor cells, hematopoietic stem cells, certain stages and types of dendritic cells, and other immature precursor cells may have a very similar morphology to ‘mature, well-differentiated, small, resting lymphocytes'. 

Intermediate to large lymphocytes range in size from slightly larger than small lymphocytes to the size of neutrophils.  The nuclei still takes up the majority of the cell, however more abundant cytoplasm is visible in these cells.  Often, the nuclei is placed eccentrically within the cytoplasm.  The nuclear chromatin is finely clumped to granular.  Typically, nucleoli are not seen although strands of loosely clumped nuclear chromatin may be mistaken for nucleoli.  The cytoplasm is lightly basophilic in color.  Occasionally these cells contain azurophilic granules suggestive of a natural killer (NK) phenotype.

Lymphoblasts are as large as a neutrophil or larger.  Size alone does not indicate neoplasia.  Very large lymphoblasts (2-4x the size of neutrophils) may be seen in reactive and hyperplastic processes.  Lymphoblasts contain round to oval nuclei with fine or stippled chromatin (loosely aggregated chromatin).  One or more nucleoli may be visible.  The cytoplasm is moderately to deeply basophilic.  Occasionally (seen more in cats than dogs) the cytoplasm may contain punctate vacuoles. 

Reactive lymphocytes are similar in morphology to small lymphocytes but are slightly larger and have more abundant, more basophilic cytoplasm.

Plasma cells are intermediate sized cells that contain small, round, eccentrically placed nuclei with condensed chromatin.  Cytoplasm is abundant, deeply basophilic, and often contains a prominent, eccentric, perinuclear, clear zone that corresponds to the Golgi. 

“Diagnosis by typical findings”

Most maturation charts show lymphocyte development as starting at the lymphoblast stage.  Cells then become progressively smaller with a more condensed chromatin pattern as they mature.  Thus lymphoblasts become intermediate cells which transition into small ‘mature' lymphocytes.  However more detailed immunologic analysis into the phenotype and structure of activated, resting, memory, effector, regulatory, and precursor lymphocytes suggests substantial overlap in morphologic features between these categories.  Morphologic features and a knowledge of ‘typical findings' has been used by both clinical and anatomic pathologists to help characterize underlying cell type and the disease process.  Some of these are described below.

Morphologic features and typical findings used to characterize atypical lymphocytes

Lymphoglandular bodies are round, homogeneous, basophilic structures comprised of cytoplasmic fragments.  The presence of lymphoglandular bodies is seen in cytologic preparations of lymphoid tissue that contains increased numbers of lymphoblasts.  This can be due to neoplasia (lymphoma) or hyperplasia.  

The presence of an eccentric, perinuclear clearing zone is often suggested as a feature of B-cells and plasma cells.  The clearing zone is the Golgi and it is a prominent feature in plasma cells.  However, the Golgi apparatus is an organelle found in most cells, including T-cells and myeloid cells. 

Sezary cells are described as medium to large lymphocytes with ceribriform nuclei.  In humans, these neoplastic T-cells are characteristic features of Sezary syndrome which encompasses mycosis fungoides, an epitheliotropic variant of cutaneous lymphoma.  A similar syndrome occurs in dogs but has been rarely reported in cats.  In dogs, epitheliotropic T-cell lymphoma is also seen in the gastrointestinal tract.  T-cells predominate in both the cutaneous and GI variants.  Interestingly, expression of protein gene product 9.5 (PGP 9.5), a marker previously considered specific for neural and neuroendocrine tissues, was recently detected in over 8/14 cases of canine cutaneous mycosis fungoides suggesting that there may be other biologic differences between the human and canine variants.

In humans, the presence of flower cells or cloverleaf cells is most often associated with T-cell disease and is particularly a feature of infection with human T-lymphotrophic virus-1 (HTLV-1).  Although perhaps more common in T cell disorders of dogs and cats, similar morphology has been seen in both B-cell and T-cell lymphoproliferative disease as well as myeloproliferative disease. 

Chronic lymphocytic leukemia (CLL)

Unlike that seen in humans where CLL is considered a disease of B-cells, CLL of dogs and cats is primarily a T-cell disease.  In dogs, CD8+ (cytotoxic) CLL predominate while in cats, CD4+ (T-helper) CLL is more common.  However, there is variation in the disease in both dogs and cats and B-cell, CD4+ T-cell, and CD8+ T-cell CLL have all been diagnosed in small animals.

In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis.

Diagnosis by flow cytometry

Flow cytometric analysis is used to define cells through a panel of phenotypic markers and receptors (usually surface) and provide a more objective characterization of these abnormal lymphocyte populations.  Lymphocyte phenotyping by flow cytometric analysis has become an established diagnostic assay for assessment of abnormal hematopoietic populations in small animal patients.  In both dogs and cats, a number of well-characterized antibodies are available for evaluation of lymphoid populations with fewer antibodies available for examination of histiocytic, myeloid, erythroid, and megakaryocytic cells.  Antibodies commonly used for flow cytometric analysis of canine and feline hematopoietic neoplasia include:

Stem cell markers

• CD34. Does not differentiate myeloid versus lymphoid origin but can be used to help differentiate acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) from more differentiated neoplasia such as chronic lymphocytic leukemia (CLL), the leukemic phase of lymphoma, or chronic myelogenous leukemia (CML).

• CD117 is a transmembrane tyrosine kinase growth factor receptor expressed on some stem cells and mast cells.

• CD172.  Found on bone marrow progenitor cells and is expressed on early B-cells (lost with the rearrangement of Ig heavy chain genes).

Hematopoietic markers

• CD45.  CD45 is a pan-hematopoietic marker with several isoforms and is found on all hematopoietic cells (including TVT) except erythrocytes.  It is expressed on lymphoid as well as myeloid precursors and mature cells.  CD45 intensity coupled with side scatter can be used to subgate cells.  Mature lymphocytes have the brightest expression of CD45 while normal lymphoblasts have much dimmer expression and neoplastic lymphocytes can lose CD45 expression entirely. 

B cell markers

• B cells express a variety of markers as they mature.  Pre-B cells, which are defined as the stage of maturation before heavy and light chain antigen receptor rearrangement, express CD79a and IgM in the cytoplasm.  As cytoplasmic IgM is exported to the surface, pre-B cells transition into immature B cells.  CD21 and surface IgM are first found on immature, naive B-cells as they enter circulation.  Following antigen stimulation, B-cells will mature and express surface IgG.

• CD79a is the transmembrane signal transduction portion of the B cell receptor.  It is found on all stages of B cells.  However, the commercially available antibody for use in dogs detects the intracellular portion limiting its utility in flow cytometry (cells need to be permeabilized for detection).  CD79a also has aberrant binding patterns which are best seen by immunohistochemistry or immunocytochemistry.  Nuclear staining is a common artifact and does not indicate B-cell origin.  Visualization of cytoplasmic positivity is necessary to confirm B-cell lineage.

• CD21 is one of the complement receptors.  With surface Ig, CD79a, CD79b, CD19, and CD35, it complexes with the B cell antigen receptor for signaling.  CD21 is expressed on mature B cells , however it may also be expressed on follicular dendritic cells in the lymph node germinal center. 

• Surface IgM is found on immature B-cells while surface IgG is found on mature B cells.

T cell markers

• Like B cells, T cells also express a variety of markers as they mature.  Cells from the thymus may give rise to thymocytes, several subsets of T-cells, natural killer (NK) cells, and dendritic cells.  Early T-cell progenitors in the thymus (thymocytes) express CD3, CD2, CD5, CD7, and CD1a.  T-cell precursors undergo rearrangement of the T-cell receptor (TCR) and begin to express both CD4 and CD8 (double positive cells).  As the cells mature further, expression of only CD4 or CD8 is maintained (single positive cells).

• CD3 is a complex transmembrane protein expressed on early thymocytes and maintained throughout lymphocyte maturation.  Therefore, CD3 is the most useful and consistent marker for detecting T cells.  However some antibodies used in flow cytometry detect part of the surface chains of CD3, while those used in IHC/ICC detect the intracellular portion of the CD3 chains.  This can lead to discrepancies in results if the neoplastic cells do not maintain the CD3/TCR complex on the cell surface.  CD3 is closely associated with the TCR and is critical for cellular activation.  The TCR exists as two forms, an alpha/beta TCR or a gamma/delta TCR.  Detection of CD3 does not distinguish whether cells are expressing alpha/beta or gamma/delta TCRs.  In the dog, monoclonal antibodies to differentiate alpha/beta and gamma/delta TCR are available. 

• CD5. Although CD5 is typically considered a T-cell marker, CD5 is expressed on mature T-cells, thymocytes, and a subset of B cells.  CD5 helps to modulate signaling through both the TCR and B-cell receptor complex by acting as a co-stimulatory molecules.  CD5 is useful in humans, because the subpopulation of B-cells that express CD5 are frequently the same cells that develop into CLL.   However, in dogs and cats, CLL is a T-cell disease and the majority of cells (both normal and neoplastic) found in blood, bone marrow, spleen, and lymph nodes that express CD5 are T-cells.  We have found several cases where only CD3 or CD5 is expressed on the surface of neoplastic T-cell populations submitted for flow cytometric analysis and routinely run both antibodies (in the same tube as CD21) to detect the overall cell phenotype. 

• Thy1 or CD90 was first described as a thymic antigen.  Expressed by most lymphocytes, but is also expressed by monocytes and macrophages.

• CD4 is expressed on helper T-cells of dogs and cats.  However, CD4 expression is also seen on canine and feline neutrophils (depending on the antibody clone used).  The CD4 seen on neutrophils is very bright while that detected on lymphocytes is intermediate to bright.  CD4 expression can also be upregulated on antigen presenting cells (monocytes, macrophages, dendritic cells).  In these cases, expression tends to be dim to intermediate.

• CD8 is expressed on cytotoxic and suppressor T-cells.  The CD8 molecule is comprised of two chains.  Alpha/beta heterodimers predominate in both dogs and cats, however alpha/alpha homodimers may also be detected in both species and may expand in certain disease.  A subset of NK cells also expresses CD8 (alpha/alpha).

• CD4CD8 double positive cells.  The presence of double positive cells is uncommon in dogs and cats although small numbers may be seen with certain inflammatory/infectious diseases (e.g. FIV of cats is the best described).  Large numbers of double positive cells suggests expansion of thymic intermediates, such as would be seen with a thymoma.

NK markers

• There are no definitive markers of NK cells in dogs.  In cats, antibodies against CD56 and CD57 can identify NK cells, although CD57 needs to be used in conjunction with CD5 and CD8 to differentiate CD8 subsets. 

Myelo-monocytic markers

• CD18 complexes with one of four different alpha subunits to form the beta2 integrins, one type of leukocytes adhesion molecules.  CD18 is found on most leukocytes, however the CD11 subunits are specific to different cell types.  CD11a/CD18 is found on all leukocytes.  CD11b/CD18 is found on granulocytes, monocytes, and some macrophages.   CD11c/CD18 is found on granulocytes, monocytes, and dendritic cells.  CD11d/CD18 is found on macrophages, splenic red pulp T-cells, and large granular lymphocytes.  

• CD14 labels monocytes and macrophages.  Not all neoplastic histiocytes label with CD14. 

• Mac387 is an anti-human macrophage antibody that reacts in dogs and cats.  It stains macrophages and monocytes, but also labels canine neutrophils. 

• Myeloperoxidase (MPO).  Cross reactive antibodies for canine (and perhaps feline) MPO have recently been identified.  In dogs, MPO labels neutrophils but also a small proportion of monocytes.

Other

MHC II is expressed on monocytes and macrophages and other APC as expected.  However, MHC II is also expressed on activated canine and feline lymphocytes.

Mast cells See CD117.

Diagnosis based on clonality

Identification of T-cell or B-cell clonality in dogs and cats requires detection of receptor gene rearrangement.  This is most typically done by PCR and is referred to as PCR for antigen rearrangement (PARR).  As part of their development, T-cells undergo rearrangement of genes encoding the T-cell receptor (TCR) while B-cells undergo rearrangement of genes encoding the immunoglobulin (Ig) receptor.  The result is that nearly every lymphocyte in the body has a unique TCR or Ig receptor.  PARR of normal tissue detects a smear or ladder of PCR products representing the diversity of the normal receptors.  Because neoplastic transformation typically occurs after the cells have undergone receptor rearrangement, all malignant daughter cells will have the same antigen receptor gene.  This is detected on PCR as a single band and represents a monoclonal population.  Occasionally bi or tri-clonal populations may also be detected. 

Canine CLL

In dogs, CLL appears to be primarily a T-cell disease although B-cell CLL has also been reported (3:1 ratio of T:B).  The cytologic morphology of T-cell CLL in dogs is typically of granular lymphocytes.  The granular cells primarily express CD3, CD8, and CD11d.  Detection of the alpha/beta TCR is more common although about a third of the reported cases express the gamma/delta TCR.  Non-granular T-cell CLL more commonly express the alpha/beta TCR but may be either CD4 or CD8 positive.  B-cell CLL in dogs express CD21 and/or CD79a.  The majority (~95% of those examined), also express CD1c.  CD5 (which is commonly seen in human B-cell CLL) is not detected on canine B-cell CLL.  B-cell CLL and T-cell CLL appear to have somewhat different patterns of disease progression.  B-cell CLL affects the bone marrow early in disease and may be considered a primary bone marrow disease.  T-cell CLL typically does not affect the bone marrow until late in the disease and may spread from the marrow after splenic involvement. 

Feline CLL

Similar to that seen in dogs, feline CLL is primarily a T-cell disease.  However, unlike that seen in dogs, feline CLL is primarily a result of CD4 or helper T-cell proliferation although occasional cases of CD8+, CD4CD8 double positive, and CD4CD8 double negative CLL have also been reported. 

Acute leukemia

While several markers are useful for the diagnosis of AML in people, AML in dogs and cats is difficult to diagnose through flow cytometry immunophenotyping as the available antibody panels do not provide lineage specific myeloid or myelomonocytic markers.  Combinations of antibodies, coupled with flow scatter patterns can be used to help characterize myeloid precursors, no currently available panel of antibodies can consistently identify myelomonocytic leukemia or AML.  The best marker for AML is myeloperoxidase.  Currently, this is detected by most labs through cytochemistry on cytology or histology slides.

Acute leukemia in the dog appears slightly more likely to be of myeloid than lymphoid origin, and about 10% of acute leukemias lack identifiable differentiation markers (acute undifferentiated leukemia).  Acute lymphoid leukemia in the dog may be comprised of cells of B, T, or NK origin. 

When examined, CD34 is often detected on AML and ALL, but is rarely (or not) detected on lymphoma and therefore serves as a useful marker to differentiate ALL with tissue involvement from lymphoma with marked leukemia.