—  SHORT COURSE #39  —

Neoplastic Disorders Of The Spleen

Section 2 - The Normal Spleen

Attilio Orazi
Dennis P. O'Malley


Histology

The diagnostic characterization of splenic disorders can be best understood in the light of the structure and function of the spleen [1, 2, 3, 4, 5, 6, 7, 8, 9]. The spleen is composed of two anatomically and functionally distinct regions [Table 1-1]. The lymphoid tissue of the spleen, called the white pulp, can be seen grossly as uniformly distributed white nodules. The white pulp of the spleen is intimately associated with the splenic arterial circulation. The central arteries, which arise from trabecular arteries within the fibrous trabeculae, are surrounded by cylindrical cuffs of lymphocytes, which are termed periarteriolar lymphoid sheaths (PALS). The PALS contain an admixture of B and T cells with a predominance of CD4 positive T lymphocytes. Periodically, splenic lymphoid follicles (Malpighian corpuscles) occur as outgrowths of the PALS [5, 6]. The morphology of the splenic white pulp varies with the age of the patient and with the presence of antigenic stimulation. Inactive or hypoplastic white pulp, in which no germinal centers are seen, is characteristic of infancy, senescence, and of the immunologically unstimulated adult spleen. In the immunologically activated state, the splenic lymphoid follicle shows three distinct zones [1, 2, 3, 7, 9]. The germinal center, composed of more numerous B lymphocytes and rare T lymphocytes embedded in a meshwork of dendritic reticulum cells, which is surrounded by a darker rim of small lymphocytes called the mantle zone, which also is composed predominantly of B cells. The mantle zone is encased by the outer, marginal zone, a cellular layer at the interface between white and red pulp. The marginal zone, composed of both B and T cells [5], is the site of antigen trapping and processing.

The red pulp of the spleen is largely composed of splenic vascular sinuses and the cords of Billroth [1, 3, 9]. The structure of the splenic vascular sinuses provides the mechanism for filtration of the peripheral blood by the spleen. The sinus lining cells, also known as littoral cells, have long cytoplasmic processes that overlap and are closely opposed. However, since no tight junctions are present, circulating blood cells are able to squeeze through the inter-endothelial spaces and percolate through the cords of Billroth before entering the splenic venous system and returning to the systemic circulation. The ability of circulating blood cells to enter the splenic sinuses and subsequently percolate through the cords depends on their deformability. Cells without the ability to deform are not able to enter the sinuses and are destroyed in the acidotic, hypoxic environment of the cords of Billroth [2].

The T-cells found in the red pulp are predominantly CD8-positive small lymphocytes which are rarely found in the PALS and are virtually absent in the germinal centers. The distribution of immunoglobulin-containing B cells is comparable to that seen in the lymph nodes. The mantle zone B cells bear surface immunoglobulin, which in most cells are IgM and IgD. The marginal zone B cells express predominantly IgM, with only a small minority expressing IgD. IgG expression is lacking in these areas and is limited to scattered cells in the red pulp were also rare IgA-containing cells are found. The red pulp contains numerous cells of monocyte/macrophage lineage, only a few of which are found in the white pulp. Natural killer cells are found scattered throughout the red pulp and within germinal centers. The red pulp also contains granulocytes, monocytes and lymphocytes which pass transiently through the red pulp circulation.

The Supporting Stroma of the Spleen
The supporting stroma of the spleen largely consist of cells with fibroblast-like morphology also known as adventitial reticulum cells, which by the way of their dendritic processes connect red pulp structures with splenic arterials, venues, and connective tissue of the splenic trabeculae. Pericytic support cells (smooth muscle actin positive) are also present in perivascular areas of the white pulp as well as throughout the red pulp. Both follicular dendritic cell and interdigitating dendritic cells are also found in the spleen. The former within germinal centers of the white pulp, the latter distributed throughout the periarteriolar sheets.

Table 1: Normal Morphologic Compartments of Spleen
WHITE PULP Follicles
- Primary 		Composed of small nodules of mantle-type B lymphocytes (see below)
- Secondary Composed of a mixture of small, irregular B lymphocytes and large transformed cells, with intermixed dendritic cells and macrophages
Mantle zone Surrounds the germinal center. Cellularity composed predominantly of small B lymphocytes with round to irregular nuclei, condensed chromatin and scant cytoplasm.
PALS Sheaths of predominantly small T lymphocytes that surround arterioles and arteries. Other cells include larger transformed lymphocytes, NK cells, plasma cells, B cells.
RED PULP Sinusoids Lined by specialized endothelial cells with macrophage capacity. Lack a continuous basal membrane.
Cords Lie between the sinusoids and composed of extracellular space and cordal macrophages
SUPPORTING STROMA Capsule and Trabecular septa Paucicellular dense fibrous tissue. Thickened in reactive/chronic conditions.

Immunohistochemical (IHC) Evaluation of the Spleen

(A list of antibodies used in our departmental lab is included in Appendix "A")

Immunohistochemistry is an invaluable tool in the evaluation of splenic disorders. It provides unique challenges, however, because of the functional complexity of the spleen and the variety of its compartments. The purpose of this section is providing general guidelines on how to evaluate the complex splenic microanatomy, stressing in particular the morphologic and the immunohistologic characteristics of the white pulp and red pulp compartments

Stroma
As a functional compartment, the stroma of the spleen is often overlooked. It does however give rise to both neoplastic and non-neoplastic proliferations which may be complex and difficult to evaluate. In addition, the stroma of the spleen may in itself, provide insight into the development of splenic disorders (e.g. extramedullary hematopoiesis).

In terms of architecture the spleen stroma can be divided into three main components: the vascular component, the reticuloendothelial or monocyte/macrophage component, and the remainder. This remaining portion consists of various mesenchymal cells and extracellular matrix which forms the structural framework of the spleen.

Vascular
The large arteries, arterioles, and veins are similar to other sites in the body, and can be evaluated with the typical battery of stains used in other sites i.e. CD34, CD31, Factor VIII (von Willebrand factor). As in other sites, neoplasms that arise from 'typical' vascular components may also be evaluated with these same stains.

The unique vascular component of the spleen is the littoral cell and its corresponding neoplastic proliferations. Littoral cells line the vascular sinuses of the spleen. The littoral cells share features of both endothelial cells and monocyte/macrophages. They are usually flat, inconspicuous cells along the edges of sinuses. When activated, they become plump and appear larger. Some of the interesting features of the cells are that they are strongly and uniformly positive for CD8, a marker which is more commonly associated with cytotoxic or suppressor T-cells. Therefore CD8 is a useful stain in delineating the sinusoidal architecture in the spleen; however, the strong reactivity with CD8 can make evaluation of CD8 positive T cells somewhat more difficult.

Monocyte/Macrophage and Dendritic Cells
The monocyte/macrophage system (MMS) is one of the more important anatomic and functional components of the spleen, forms the bulk of the red pulp (cordal macrophages), but often receives little attention in its overall evaluation. Only when there is a significant derangement of the MMS, is it even noticed. In its normal function, the MMS serves as a functional filter for effete red blood cells (RBCs) and other less than desirable components of the circulating blood. In conditions in which there is an increase in destruction of circulating blood components, as in an autoimmune hemolytic anemia or immune thrombocytopenic purpura, the cordal macrophages will become engorged and hyperplastic within the spleen producing red pulp congestion. The cordal macrophages can also become engorged with metabolic products in storage diseases, infectious agents or even abnormally produced immunoglobulins.

Evaluation of the MMS using IHC can be accomplished by a number of antibodies which include CD68, lysozyme, anti-chymotrypsin, and others. When there is accumulation of intracellular material in macrophages, histologic stains may be of considerable value, depending on the accumulated product. Most abnormal storage proteins are PAS positive. Infectious organisms can be evaluated by the judicious use of AFB, GMS, tissue Gram and PAS stains. In the case of immunoglobulin accumulation, IHC for kappa and lambda light chains may be of benefit.

Dendritic Cells
A considerable portion of the spleen (white pulp) is devoted to its function as a lymphoid organ. Components that are notable parts of this are dendritic cells. The two major types of dendritic cells are interdigitating dendritic cells (IDC) predominantly located in the T-cell areas and follicular dendritic cells (FDC), predominantly located in the follicles. FDC play an integral role in B cell maturation, antigen presentation and clonal selection within the follicle.

Distinction of dendritic cells is usually accomplished by immunohistochemistry. Dendritic cells are varyingly positive for monocyte/macrophage-associated markers such as CD68, S100, CD1a, lysozyme and alpha-1-antitrypsin. FDC can be reliably distinguished from IDC by application of immunohistochemical stains for CD21 and/or CD35. Ultrastructural examination of the FDC reveals the presence of desmosomes, distinguishing the process from IDC, which lack this type of cell junction.

Other Stromal Elements
Evaluation of structural mesenchymal cells and extracellular matrix (ECM) by immunohistochemistry can be performed using various antibodies. Low-affinity nerve growth factor receptor (LNGFR; ME20-4) identifies reticulum cells in the splenic cords, periarteriolar and pericapillary adventitial cells as well as follicular dendritic cells. The LNGFR positive cell subsets all share a fibroblastic or spindle cell morphology and perform mainly structural functions. Smooth muscle actin (SMA; 1A4) can be used to identify pericytes and cells with myofibroblastic differentiation (myoid cells). SMA staining highlights a concentric reticular meshwork of SMA positive cells present at the interface between red pulp and marginal zones. SMA staining of perivascular cells within the red pulp can also be observed. The ECM can be evaluated by immunohistochemistry for collagen IV (CIV22; basement membrane type collagen) and by reticulin staining. Collagen IV highlights the "ring fibers" which incompletely surround the littoral cells of the splenic sinusoids. Other types of collagen are collectively stained by Gomori silver impregnation technique also known as reticulin stain. Various patterns of stromal cell hyperplasia or alterations in the architectural distribution of ECM proteins can be observed in a variety of splenic disorders [7].

Lymphoid Compartments
In evaluating lymphoid disorders of the spleen, much of the knowledge of lymph node pathology can be directly translated. Most of the proliferations that are confined to the white pulp share morphologic and immunophenotypic characteristics with their lymph nodal counterparts. Some of the difficulties arise in lymphoid proliferation that are unique to the spleen and lymphoid proliferations that have a significant red pulp involvement. In parallel with our understanding of phenotypic and immunologic composition of the hematopoietic system, our armamentarium of paraffin section-reactive markers has grown as well. In some cases, it could be said that we have too many markers, leading to confusion and challenge in choosing and interpreting them wisely.

B and T Cell Markers

CD3/CD20
In lymphoid lesions, this pair should usually be always used in concert [11, 12, 13]. This allows assessment of number and distribution of B-cells and T–cells. It is important not to order only a CD20 stain alone, because the number of T-cells can be significantly underestimated. CD45RB, CD45RA and CD45RO are generally not 'first-line' choices. If the lesion is morphologically lymphoid, then these stains add no diagnostic information in the evaluation once a CD20 and CD3 have been performed.

CD45
The use of CD45 is largely limited to the confirmation of the hematologic nature of a cell proliferation. If lymphoma is a diagnostic consideration, it may be added to the CD3/CD20 combination, as mentioned above. If other hematopoietic lesions (ex. myeloid sarcoma, plasmacytoma) are being considered, then specific stains (e.g. myeloperoxidase – see below) should be used. CD45 is often useful in the context of classical Hodgkin lymphoma as a negative finding.

CD43
The addition of CD43 to the CD3/CD20 combination adds considerable value to the diagnostic combination [14, 15]. CD43 is expressed on a variety of normal hematopoietic cells including; normal T-cells, most marrow-derived cells, and macrophages/histiocytes. It is not typically co-expressed on normal B-cells, while is often expressed by malignant B-cells, including several subtypes of small B-cell lymphoma. CD43 is almost always expressed on mantle cell lymphoma (MCL) and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). It is also expressed in a subset of marginal zone lymphoma (MZL) lymphoma (20-40%) and is essentially never expressed in follicular lymphoma (FL). In essence, CD43 is useful in several ways: 1) if positive in B-cells, then it is almost always lymphoma 2) it is a confirmatory stain for T-cells and should parallel CD3 staining 3) it can help identify hematopoietic neoplasms that are CD3- and CD20-, such as myeloid sarcoma, mast cell disease and plasma cell neoplasms.

CD15/CD30/CD45/Pax-5
This combination of stains is valuable when evaluating classical Hodgkin lymphoma (HL) as a differential diagnosis (see section on cHL for further discussion). CD45 positivity in single, large neoplastic cells greatly decreases the likelihood of cHL and favors other diagnostic considerations. CD15 positivity in large cells supports a diagnosis of cHL. However, the lack of CD15 should not prevent the diagnosis. Finally, CD30 has become a very important tool in the hematopathology toolkit. It should be strongly, but variably positive in cHL in the majority of tumor cells. Also, it should be strong and uniformly positive in cases of anaplastic large cell lymphoma (ALCL). In difficult cases, the positivity of cHL with Pax-5 and its negativity with Alk-1 may help in the differential diagnosis between cHL and ALCL.

BCL-2
A common consideration in spleen pathology is the differential diagnosis of FL versus follicular hyperplasia. Although there are several morphologic criteria that are valuable, immunohistochemical confirmation is always reassuring. Bcl-2 is an anti-apoptosis protein that is expressed in a variety of cells. In normal cells of the follicle center, it is not expressed, as benign germinal center cells need the capacity to undergo apoptosis as a function of clonal selection. In benign follicles, bcl-2 protein is positive in the mantle zone cells but negative in follicle centers. Conversely, in FL, the B-cells of the follicle are positive for bcl-2 in about 75% of cases. It should be remembered that this leaves 25% that lack bcl-2 positivity. Also, many normal cells express bcl-2 protein including benign mantle cells and most T-cells. It is important to remember that bcl-2 does not help distinguish between different types of B-cell lymphomas. About 75% of all B-cell lymphomas express bcl-2, including most cases of MCL, SLL/CLL and MZL. Other stains that can be used to confirm follicular origin of cells include Bcl-6 and CD10.

TRAP and DBA-44
Several antibodies are available for the evaluation of hairy cell leukemia (HCL) in the spleen. Flow cytometric findings are often distinctive (typically positive for CD11c, CD22, CD25, and CD103). IHC is particularly useful when flow analysis is not available. The main differential diagnosis includes the area of overlap between HCL and splenic marginal zone lymphoma (SMZL). Upon establishing a B-cell proliferation through the use of CD20, the most powerful support of a diagnosis of HCL is the presence of positive staining for TRAP by immunohistochemistry. DBA-44 is supportive, but can also be positive in a proportion of SMZL.

Other Markers
Additional immunohistochemical stains for the diagnosis of lymphoma include a lengthy list of markers. Only selected stains will be discussed further. ALK-1/p80 is the gene product of the anaplastic lymphoma kinase gene. It is overexpressed in the commonest subset of ALCL, and can be used to confirm the diagnosis. It is very rarely expressed in a subset of diffuse large B-cell lymphomas. Cyclin D1 is expressed in most (but not all) cases of mantle cell lymphoma. It is also expressed by many non-hematopoietic neoplasms and normal cells, and is rarely expressed by hairy cell leukemia and plasma cell myeloma.

Additional B-cell Markers
Adjuncts used to identify B-cells are helpful in cases that do not express CD20, or express it only weakly, such as CLL/SLL or B-cell acute lymphoblastic leukemia (B-ALL). In these cases, the most useful antibodies are probably CD79a and Pax-5, both reliable pan-B markers. Besides its normal expression in T-cells, CD5 can be used to evaluate several small B-cell proliferations. CD5 is coexpressed on CLL/SLL and MCL and only rarely on other B-cell lymphomas. In conjunction with CD23, CD5 can be used to differentiate small B-cell lymphomas. MCL would be CD5+/CD23-, CLL/SLL would be CD5+/CD23+ and virtually all cases of FL or MZL are negative for both CD5 and CD23.

Additional T-cell Markers
Occasionally, additional T-cell markers are useful in the evaluation of T-cell lymphomas. Besides CD3 (and CD43), as pan T-cell markers, CD2, CD5 and CD7 are expressed early in T-cell differentiation and throughout the life of T cells. Loss of pan-T-cell markers is common in T-cell lymphomas, and can support this diagnosis.

CD4 and CD8 are markers of T-cell differentiation. As mentioned earlier (see VASCULAR), CD8 interpretation in the spleen is complicated by the positivity seen in splenic littoral cells, the cells lining splenic sinuses. Most peripheral T-cell lymphomas express CD4. However, there are occasional CD8-positive lymphomas, as will be discussed later. Some lymphomas, including precursor T cell neoplasms, can be either 'double-positive' (CD4+CD8+) or double negative (CD4-CD8-).

Two neoplasms of T-cells require particular attention in evaluation of splenic pathology: large granular lymphocyte leukemia (LGL) and hepatosplenic T-cell lymphoma (HSTCL). LGL disease can be derived from cytotoxic CD8+ T-cells, from NK-like cytotoxic T-cells or from true NK cells. By conventional immunohistochemistry, the distinction is almost impossible, although flow analysis would clarify the issue. These cells are typically positive for CD3 (NK cells have cytoplasmic positivity, so IHC would be positive), CD8 (in T-cell type) and CD56 (in NK and NK-like). HSTCL is of T-cell lineage, but has a somewhat characteristic immunophenotype: its cells are positive for CD3 and most often negative for CD4 and CD8. They will often express CD56.

Both HSTCL, LGL and other subsets of T-cell lymphomas will stain positively with markers for cytotoxic molecules such as TIA-1. Perforin and granzyme B can also be used to identify proliferations of cytotoxic T cells and NK cells; these markers are also variably positive in granulocytes and myeloid precursors.

Ki-67
The use of Ki-67 (MIB1) as a diagnostic aid comes as little surprise to most. It has long been used as a prognostic marker in a variety of pathologic conditions. Its applications as a tool to identify specific diagnostic entities, has been applied less strictly and little has been published on this topic. The essence of its value lies in comparing the proliferative characteristics of normal cell components to their abnormal counterparts. If a compartment of the spleen is typically highly proliferative (e.g. germinal center), then a low proliferation rate may indicate the presence of abnormal cells. In this way, it may be possible to distinguish between follicular hyperplasia (high Ki67 staining) and a low grade follicular lymphoma (low Ki67 staining). It may also be useful to note that many low grade lymphomas have very low proliferation rates and may be detected by their disruption of normal patterns of proliferation. Similarly, increased proliferation distributed in the red pulp may indicate the presence of neoplastic cells that may be indistinct by other methods of evaluation.

Specific Infectious/Viral Stains
Viral reactions can sometimes simulate lymphomas. Immunohistochemical stains for Herpes virus (Type I and II), CMV and EBV (EBV-LMP) can often avoid misinterpretation of such lymphoma mimics. EBV-LMP (or the EBER in situ technique for EBV-RNA) can also be used in support of a diagnosis of cHL (particularly mixed cellularity type) versus ALCL or T-cell rich B-cell lymphoma, which are typically negative.

Immunohistochemical Evaluation of Myeloid and Histiocytic Disorders in the Spleen

Anti-myeloperoxidase (MPO) /CD34/ CD68 (PGM1 versus other)
In most cases beyond early infancy, erythroid and myeloid precursor cells are not found in the spleen in any significant number. Rare megakaryocytes are, however, normally seen. When there is an accumulation of marrow derived cells in the spleen, this is commonly referred to as splenic myeloid metaplasia or extramedullary hematopoiesis (EMH).

EMH can be seen in a variety of both neoplastic and non-neoplastic conditions. EMH is not uncommonly seen in spleens removed for reactive pathologic conditions and is in most cases histologically benign. However, it can be of great importance to identify cases associated with neoplastic EMH in the spleen. The clonal marrow diseases associated with neoplastic EMH include myeloproliferative (MPD), myelodysplastic (MDS), and combined MPD/MDS disorders, as well as acute myeloid leukemias.

Red cell precursors, with their perfectly round, dark purple nuclei and intrasinusoidal clustering are often easier to recognize. In contrast, immature granulocytic cells, for example myelocytes, can be difficult to distinguish from other cell types present in a hyperplastic red pulp. Megakaryocytes are the most easy to recognize, based on their size and nuclear lobation. However, abnormal forms (e.g. micromegakaryocytes) are best identified by using anti megakaryocyte antibodies.

The stains used to identify EMH and its components in marrow are the same stains used for bone marrow biopsies. For granulocytic or monocytic cells the stains include: MPO, CD34 (a hematopoietic progenitor marker, also positive in non-sinusoidal vascular cells), CD117 (c-kit; also positive in mast cells), and CD68 (PGM-1 is preferred to KP-1 stain for its increased specificity). For erythroid precursors, both hemoglobin and glycophorin stains work well. For megakaryocytes, CD42b or CD61 are quite useful, with CD41 and Factor VIII also serving this purpose.

The presence of clusters of cells positive for early precursor-associated antigens (CD34 or CD117) in the spleen of a patient with an established, chronic marrow disorder may indicate extramedullary acute transformation or acceleration of a previously stable MPD.

Systemic mast cell disease commonly involves the spleen. The IHC evaluation of mast cell disease can be very helpful in establishing this often elusive diagnosis. The single most specific and sensitive stain for the evaluation of mast cell disease is an IHC stain for tryptase. This will highlight the mast cells.

Other stains that are positive in mast cells (both benign and neoplastic) include: CD117, CD2, CD43, and CD68. It has been noted recently that CD25 expression in mast cells seems to correlate with neoplastic rather than reactive mast cell lesions, although further studies are warranted. Chloracetate esterase and Giemsa stains can also be occasionally useful, and this is true of the even less frequently-used toluidine blue stain as well.

References
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  15. Segal GH, Stoler MH, Tubbs RR. The "CD43 only" phenotype. An aberrant, nonspecific immunophenotype requiring comprehensive analysis for lineage resolution. Am J Clin Pathol. 1992 Jun;97(6):861-5.

  16. Poster or Platform Presentation: Orazi A, O'Malley DP, Thomas JL and Czader M. Abstract: Stromal changes in reactive and malignant disorders of the spleen. United States and Canadian Academy of Pathology Annual Meeting, Vancouver , British Columbia . March 6-12, 2004.