Outer Mantle

Trisomy 12 del 13q t(2;X4) t{2;18) t(14;19) t(18;22)




classification and immunophenotype in low grade B-cell NHL



CDS and CD23


Figure 2. The lymphoid system (major organs and tissues)

Figure 3. B-lymphocyte trafficking

lymphocytes are highly controlled and not just a series of random stochastic events and how it occurs is now leading to a fundamental change in our understanding of the behaviour of the malignant counterparts of these cells10. It is becoming clear that several shared ligands and their receptors are responsible for controlling the circulation and migration of lymphocytes - particularly the integrms (Table 4) and the selectins and some members of the immunoglobulin superfamily (Table 5).


Lymphocytes are derived from haemopoietic stem cells in the bone marrow and the stages of development of the B-cells can be broadly divided into three phases - Lymphopoiesis (non antigen driven) in the bone marrow, immunopoiesis (antigen driven) in the lymph nodes followed by release into the circulation with homing to MALT and other areas (12) and to sites of inflammation (Figure 3). B cell development has been divided into at least six major stages of development based on expression oF B-cell receptor proteins, Ig rearrangement status -and other (CD) antigen expression-(11). The first step is the acquisition of the associated parts of the B cell receptor (CD19/CD21/CD79b) to form a 'pro-B cell' (13) - which remains anchored in the bone marrow by several adhesion molecules, particularly CD117 (c-kit), VLA-4 and VCAM-1 (14). This cell is at a stage of development before immunoglobulin gene rearrangement has taken place. The process of immunoglobulin gene rearrangement is complex involving selection of one variable (V) gene from over 300 on chromosome 14 followed by splicing to a diversity (D) gene selected from more than 30 and then selection from 6 junctional (J) genes. At each stage somatic hypermutation occurs involving deletions or additions of nucleotide bases allowing frame shifts to increase the diversity of potential immunoglobulins15. B-lineage lymphoid acute leukaemias are most commonly derived from cells at the pro-B (or earlier) stage. Gene rearrangement results in unique complementarity determining regions (CDR) and since they occur early in B-cell development act as clinically useful markers in lymphoma (where the phenotype is generally of a much more mature B-cell) both as a diagnostic tool to establish clonality and as a means of monitoring residual disease after treatment. At the pre-B cell stage the cell has not yet rearranged a light chain so only protein is expressed and is associated with a 'surrogate' light chain (Xs) for stability (16). Conventional light chain gene rearrangement follows with kappa first and if this fails then lambda resulting in an "immature B" cell. Cells that fail to rearrange their light chain genes successfully undergo apoptosis. By this time the cell is expressing a complete immunoglobulin IgM molecule on the surface which acts as a receptor for further differentiation into "mature B" cells. Mature B cells begin to co-express IgD (using the same V gene), and interact with other surface transmembrane proteins (Ig-and and enabling repetitive antigen interactions with T-cells or APC's results in intracellular signalling (via the tyrosine kinase syk) (17).

Table 2. Cytogenetic. molecular and immunophcnotypic associations in lymphoid malignancies


Table 2. Cytogenetic. molecular and immunophcnotypic associations in lymphoid malignancies




cfi+ preB ALL



CD 10 negative, CD 15+ ALL

t(9;22){q34;pl 1 )p 190


mixed phenotypes, bad prognosis



mixed phenotypes, poor prognosis



CD 13+ B-lineage ALL

TALI deletion


T lineage ALL



T lineage ALL



T lineage ALL

1 lq23-many


B lineage ALL



T lineage ALL




t(8;22)(q24;q1 1)








C D5-/C D23+/CD10+ FL



CD5+/CD23- MCL



ALCL (T lineage)



Marginal Zone Lymphoma (MALT)






Benign polyclonal B-cell


Trisomy 12


proliferation CD5+ CD38+ 'postfollicular' CLL CD5+ CD38W 'prefollicular' CLL

Key: ALL=Acute Lymphoblastic Leukaemia/Lymphoblastic Lymphoma

MCL = Mantle Cell Lymphoma ALCL=Anaplastic Large Cell Lymphoma FL = Follicle Cell Lymphoma MALT = Mucosa Associated Lymphoid Tissue CLL = Chronic Lymphocytic Leukaemia

Table 3. CD numbers in lymphoma diagnosis

CD Nos Roles in diagnosis

CD 1 a Cortical thymocyte marker, present only at a transient stage of T cell development in the thymus. The prcscncc of CDla+ cclls in the peripheral blood or bone marrow usually suggests a T-cell at the lymptioblast stage (eg T-all/lymphoblastic lymphoma). CD 2 T cell marker which appears very early in T cell development. Expressed on peripheral T cell lymphomas and Natural Killer cell malignancies CD 3 Pan-T ccll marker associated with the T ccll antigen reeeptor. CD3 appears in the cytoplasm of the cell earlier than on the surface, and cases of T-ALL are usually surface CD3 negative but cytoplasmic CD3+. CytCD3 is therefore a useful and the specific marker of T cell lineage in NHL. CD 4 Present on the helper subset of T lymphocytes. It is also found on normal monocytes.

Present on Sezary/Mycosis Fungoides cells CD 5 Pan-T cell antigen which is expressed early in T cell development and on a subset of normal B cells, the malignant expansion of which gives rise to B-CLL. Strongly expressed in (B) mantle cell NHL and in T-NHL.. CD 7 Pan-T ccll antigen which appears early in T celll development, and is also present on

Natural Killer cells. The antigen is expressed quite weakly on normal mature T cells, but very strong expression is seen in T-ALL and T-PLL, whereas Sezary cells are usually weak or negative, CD 8 Found on the cytotoxic subset of T lymphocytes on most natural killer cells. Slightly waek expression is common in T-NHL. CD 10 The Common Acute Lymphoblastic Leukaemia Antigen (CALLA).

Present in many cases of follicle centre cell lymphoma (B-cel!) and in lymphoblastic lymphoma and occasionally in other high grade B-NHL CD 11 A family of cell adhesion antigens with a wide distribution on lymphoid and myeloid cells

CD11 a... present on T cells, B celts, granulocytes and monocytes

CD1 lb ... present on lymphocytes, and most granulocytes and monocytes

CDllc ... present on B cells, Natural killer cells, granulocytes, and particularly monocytes.

CD 14 Monocyte/neutrophil marker. Monocytes express CD14 strongly. It is useful in assigning monocytic lineage eg Langerhans ccll tumours. CD 15 A myeloid marker which is strongly expressed on most mature neutrophils, and weakly expressed on monocytes. It is also found on Reed-Sternberg cells (Hodgkin's Lymphoma)

CD 16 This marker is found on most mature neutrophils and may also be found on Natural Killer cells malignancies

CD 19 A reliable B cell marker which appears early in B cell development and persists until the terminal plasma cell stage, where it is usually lost. This is the most widely used B cell marker and virtually all B cell malignancies (except myeloma) arc positive CD 20 AB cell marker which appears later than CD 19 in B cell development, and is positive on all mature B ceils and most B lineage NHL CD 22 AB cell marker which appears in the cytoplasm much earlier than most other B cell markers, and is only expressed on the surfacc of relatively mature B cells.CytCD22 is therefore one of the most reliable markers of B-lineage NHL CD 23 Activation antigen, present on a subset of normal B cells, and virtually all cases of CLL and follicular NHL

Table 3 to be continued...

Table 3. continuing_

CD Nos Roles in diagnosis_

CD 25 CD25 Activation antigen, which is only very weakly expressed on activated T and B cells, but is an important marker for the diagnosis of hairy cell leukaemia (medium/strong positively, whereas hairy cell variant and splenic lymphomas (villous and non villous) are usually negative). Often expressed in peripheral T-NHL CD 30 Ki-1 antigen present in Hodgkin's Lymphoma and in anaplastic large ceil lymphomas.

It's presence in NHL may suggest a better prognosis. CD 34 Stem cell marker, present on progenitor cells of the bone marrow. An important marker to diagnose the immature subtypes of lymphoblastic lymphomas CD 38 Antigen present on activated T cells, some B cells, and monocytes. The antibody is very useful to demonstrate the presence of plasmacytic malignancies (multiple myeloma and some NHL). CD 45 Lcucocytc common antigen, present on all leucocytes, but absent from red cells and pi This is extremely useful in deciding whether a tumour is of haemopoietic lineage, haemopoietic tumours will not express this marker, but negativity can be seen in cases lymphoblastic leukaemia.

This antigen exists in several different isoforms:

CD45RA: in the peripheral blood found on 60-80% of T cells, all B cells monocytes, and granulocytes. Positive T cclls arc "naive".

CD45RB: in the peripheral blood found on 80% of T cells, all B cells, an monocytes.

CD45RO: in the peripheral blood found on 20-40% of T cells, also present on most monocytes and granulocytes, but absent from B cells. T cells expressing CD45RO have been antigenically stimulated (memory cclls). CD 52 A gpi anchored antigen recognised by CAMPATH-1 antibody, which has broad reactivity against lymphocytes and monocytes. The antibody is of benefit in treating some NHL and CLL cases CD 56 NCAM is the most important marker of natural killer cells. However it is not restricted to haemopoietic cells and is found in other tumours (eg PNET and neuroblastoma).

CD 64 High affinity receptor for IgG, mediates antibody-dependent cell-mediated cytotoxicity. Weakly expressed on activated neutrophils, with monocytes and monoblasts showing stronger staining. Useful for Langherhans cell tumours CD 79 This antigen is a dimer and is associated with the B cell immunoglobulin receptor:

CD 79a: considered to be the best specific marker of the B cell lineage, present o cells at all stages of maturation, including plasma cells. This antigen is best detected cytoplasm.

CD79b: present on most B cells, but absent on plasma cells. Also negative on cases of CLL, and can help to distinguish these from other B-cell malignancies. CD 103 This antigen is present on a very small number of CD8+ T cells in the peripheral blood, and some lymph node T cells. It is also present on hairy cells, and is an important marker to distinguish hairy cell leukaemia from splenic lymphoma with villous lymphocytes

CD 138 Syndecan-1 antigen, present on some B-cclls and expressed on most plasma cells. Ki-67 A useful marker of cell cycle activity that can be used to distinguish Burkitt'sand lymphoblastic NHL (high Ki67+) from mantle cell and high grade NHL (medium Ki67+) to low grade lymphomas (low Ki67+). Cyclin DI Another useful marker of cell cycle activity that is overexpressed in mantle ccll lymphoma which is generally regarded as diagnostic of that condition, ALK The protein product of a gene on chromosome 5 that is not normally detectable on white cells, but is overexpressed in some T-lineage high grade lymphomas, especially those associated with the t(2;5) NPM-ALK translocation. These types of anaplastic large cell lymphoma tend to occur in young people and are associated with a good _prognosis_

Table 4. Adhesion molecules involved in lymphocyte trafficking


Table 4. Adhesion molecules involved in lymphocyte trafficking



a subunit

ß subunit



al (CD49a)

I,aminin, collagen


a2 {CD 49b)

Collagen, Laminin


a3 (CD 49c)

Fibronectin, laminin,



a4 (CD 49d)

VCAM-l, fibronectin


a5 (CD 49e)



a.6 (CD 49f)








av (CD 51)



aL (CD lia)


IC AM-1,2,3


«M (CD 11b)


ICAM-1,3, fibrinogen


ax (CD 11c)

Fibinogen, C3bi


ctlib (CD 41)



Fibronectin, Fibrinogen,


av (CD 51)



a6 (CD 49f)

ß4 (CD 104)



av (C 51)


Vibronectin, fibronectin


av (CD 51)




a4 (CD 49d)


Fibronectin, VCAM-l,



av (CD 51)


Table 5. Adhesion molecules involved in lymphocyte trafficking Se lectin s



Expressed on



Main role



E-select in




Memory T

Neutrophil and



monocytes, CD4+


capture and rolling



Many WBCs



Homing of




P-se lectin





Neutrophil and



monocyte capture



and rolling




Immunoglobulin superfamily



Expressed on



Main role




Lymphocytes and many others

Lymphocytes and many others

Adhesion of WBC's to endothelium and extravasation





CD 102




CD 106

CD3 i


Lymphocytes and many others

Endothelial cells and many WBC's

Lymphocytes, neutrophils, monocytes,

Endothelial cells, and many WBC's

Endothelial cells, platelets, monocytes, granulocytes


Mucosal HEV

Lymphocytes and many others

Many WBC's

Lymphocytes and many others

Adhesion of WBC's to endothelium and extravasation

Lymphocyte trafficking

Regulation of intercellular adhesion

Lymphocytes Adhesion of WBC's Monocytes to endothelium and extravasation

L-se lectin a4 U7

Endothelial cells





Many WBC's

Extravasation and transmigration

Lymphocyte homing to MALT areas

Further differentiation (at this stage termed "antigen dependent immunopoiesis") is largely cytokine driven and takes place in the secondary lymphoid tissue (for example mesenteric lymph nodes, mucosal associated lymphoid tissue (MALT) and the spleen).

B cells are capable of directly recognising some antigens that are able to cross link the B-cell receptor (and thereby induce B cell proliferation) independently of T-cell help. These antigens (T-independent) tend to be large and repetitive in their epitope structure (e.g. pneumococcal polysaccharide). However, most antigens are not recognized by B-cells and require preliminary recognition by T-cells. The B-cell then recognises the T-cell receptor and this is followed by several ligand interactions - LFA1 (B-cell) with CD54 (T-cell) followed by CD40 (B-cell) with CD40 ligand on the T-cell. A second series of events is triggered then -LFA3 (B-cell) with CD2 (T-cell) (18). CD2 expression appears in conjunction with CD28 on T-cells, and interacts with CD80 and CD86 on APC's. These costimulatory molecules on T-cells are the key to this phase of antigen dependent B-cell further differentiation. The antigen presenting cells involved in the germinal centre are follicular dendritic cells and the B cells (by now centroblasts) proliferate rapidly (in the so called dark zone of the follicle centre). A number of important events mark the transition from centroblast to centrocyte. Somatic hypermutation in centroblasts results in a wide and continually expanding repertoire of B-cell receptors. Selection then has to take place and many are deleted at this stage. Phenotypically, follicle centre cells at the centroblast stage do not express CD44 and L-selectins, whereas centrocytes do. Cross linking of the B-cell receptors also induces CD23 expression and upregulation of HLA class II, (hence the classic follicle centre lymphoma that is

CD5-/CD23+). CD10 is also expressed by centrocytes and by this stage that immunogloblin class switching is complete.

Traditionally, lymphocyte commitment to production of a single antibody was thought to occur following a single chance encounter with correctly presented antigen (on APC) or appropriate T cell receptor (on a T cell). There is now evidence that this occurs over a period of time - the cells needing repeated encounters with antigen (multiple 'kisses'). This hones and defines both the selected cells and increases the affinity of antibody. Humoral immune responses result in a polyclonal B cell proliferation, producing antibody with varying affinity for antigen. Affinity maturation (whereby the mean affinity of the polyclonal response to an antigen increases) occurs in the germinal centre19. Many B-cells die at this stage through apoptosis, since despite successful gene rearrangement, their antibody is of too low affinity or they have too infrequent antigen contact to accomplish the events described above to go on to become memory cells or plasma cells. B-cells can also be rescued from apoptosis by interaction of ligands with follicular dendritic cells (CLA-4 (B cell) with 1CAM-1 and VCAM-1) and it is probably a combination of these events that operates the selection procedure20,21.

After this, B-cells either mature further to become memory B cells (through CD40 cross linking with CD40L on T cells in the centrocytic area of the germinal centre) or (if there is CD21/CD23 cross linking) become plasma cells.

High affinity antibody producing B cells are a feature of memory cells, a subset of naive B-cells (IgD+) derived from the centrocyte which migrate to the mantle zone of the lymph node. These memory cells acquire the antigen CDS. The function of CD5+ is currently unknown. It is clear that CD5+ B cells are derived separately from CD5' B cells22 and that the former show restricted V gene usage as a result of antigen driven selection whereas the latter utilize a wider range of V genes.

The malignant counterpart of these CD5+ memory cells is chronic lymphocytic leukaemia or lymphocytic lymphoma. However, recent work has shown two CD5+/CD23+ populations of B-cells exist, one of which recirculates back to the inner mantle zone and can also form the malignant counterpart of what is recognised as a different type of CLL.

Some naive B-cells have less avid antibody affinity yet do not undergo apoptosis. They fail to differentiate fully in the germinal centre and migrate through the mantle zone to the marginal zone where further T cell interaction and B cell selection occurs. Having completed the final stage of development beyond the centrocyte stage, mature B-cells lose their characteristic clefted nucleus and are released into the lymph and enter the blood stream via the thoracic duct. In the blood stream the highly controlled migration continues. IgA secreting cells express the integrin a407 (the mucosal homing receptor) which binds to the mucosal cell adhesion molecule (MAdCAM-1) in the gut mucosa, Peyer's patches and the mesenteric lymph nodes and then circulate back to regional lymph nodes (10). IgG, A and E producing cells in the blood, traffic to secondary lymphoid tissue. Lymphocytes enter the lymph node through specialised high endothelial venules (HEV's) found in the interfollicular areas where they come directly into contact with T cells. The process of travelling to sites of infection is directed by adhesion molecules (which bind to lymphocytes relatively loosely) with the result that lymphocytes roll along the blood vessels, directed by integrins and selectins to their destination. The destination is defined when a stop signal is encountered. These stop signals are delivered by the stronger binding cell adhesion molecules e.g. ICAM1 (CD54), ICAM2 (CD 102) and LFA1 (CD11a/CD18). The final journey to their destination is by local migration under the influence of chemoattractants.

Mature B cells express surface immunoglobulin. When antigen binds to it this usually results in proliferation of the cell and secretion of the immunoglobulin. This secreted Ig may exert a negative effect by virtue of cross linking the B cell receptor sites that are occupied with antigen (via tyrosine kinases of the ZAP70/Syk family), followed by binding to phosphotyrosine phosphates activated by SHIP/Shc binding), and this seems to be important for preventing excess production of immunoglobulin. A group of lymphoma related diseases are characterized by overproduction of immunoglobulin, such as MGUS (of IgM or IgG class) and in these conditions defective negative signalling by this mechanism has been shown and is thought to be due to competition between SH2 binding domains on the different protein messengers (17).


The development process is also highly controlled for T cells which have specific chemokine receptors to direct them. In contrast to B-cell development which occurs widely at sites of immune recognition, T-cell development is much more restricted to T-cell areas located in the paracortical regions of lymph nodes and spleen, the thymus and the skin and gut. There is another fundamental difference between T and B-cells. T-cells need to recognise peptide antigen in the context of MHC whereas B-cells are able to recognise soluble antigen directly with the Ig B-cell receptor complex. There are some exceptions to this such as the ability of some T-cells to recognise HLA alloantigens and some superantigens directly. For B-cells, direct antigen recognition is also the mechanism whereby autoreactive B-cells are taken up in the spleen and rendered functionally anergic since they are exposed directly to antigen there.

T cell precursors arise from a common lymphoid progenitor derived from the CD34+ compartment of the bone marrow. Germ line cells that are committed T cells (prothymocytes) and express cytoplasmic CD3+ and CD7+ migrate to the T-cell processing areas (thymus, skin and gut). As they mature T-cells migrate from the cortex to the medulla of the thymus. An important enzyme involved at this stage is Terminal deoxynucleotidyl transferase (TdT), which is expressed by developing T-cells in the cortex of the thymus. It is here that VDJ joining and TdT activity occurs, increasing by hypermutational events the potential repertoire of T cell receptors (TCR) to be formed. At this point, T cells express both CD4 and CD8, (common thymocytes) following which they migrate to the medullary thymic areas to become or cells

(mature thymocytes). Most mature T cells express the heterodimeric TCR

protein, but a smaller circulating, yet important category of T cells carry the TCR heterodimer. The T cells are involved in first line defence immune responses (they are thought to be evolutionally primitive) and home to mucosal sites as well as the skin. Thymocytes of the TCR lineage undergo thymic selection to ensure autoreactive T cells are deleted, and following this are released into the circulation as mature or T


T-cells account for 2/3 of the circulating lymphocytes and also have a longer life span than B cells. They circulate in a similar manner to B cells though high endothelial venules to secondary lymphoid tissue and via lymph back to the blood again.

Homing of T cells is also controlled by adhesion molecules of three main families-the selectins, the immunoglobulin superfamily, the integrins and CD44.

Another important antigen pivotal in T cell development is CD45 which exists in a number of isomeric forms at different stages of ontogeny and is hence a useful marker in looking at malignant counterparts of T-cells. Four different CD45 isoforms, recognised by monoclonal antibodies to different epitopes, are seen on T cells, ranging from 180kD to 220kD (CD45RA, CD45RB, CD45RC, and CD45RO (the lowest molecular weight one). Memory T-cells are characterised by their expression of CD45RO, L-selectin and the integrin LFA-1. LFA-1 (a! JJ2) is a cellular adhesion molecule of the integrin family. In this family there is a common B2 unit (CD 18) associated with distinct a subunits - a,(CD 11 a), aM(CD116), ax(CDllc) and otD(CDlld). LFA-1 is involved in leucocyte migration locally (at the site of inflammation) and 3 ligands have been characterised - ICAM1 (CD54), ICAM2, and ICAM-3.

Naive T-cells are CD45RA+ and also express LFA-1 which binds preferentially to the high molecular weight forms of CD45 whereas CD2 binds to the low molecular weight forms of CD45 (CD45RO). Other integrins such as VLA-4, 5 & 6 and the CD44 family are also preferentially expressed on memory T cells. Some T-cells that home to the skin do so by expression of a cutaneous lymphocyte associated antigen (CLA) that bind to a selection - E-selectin.

By differentially expressing different types and levels of these adhesion molecules, T cells can migrate selectively though the high endothelial venules encoutering different ligands at different HEV sites such as MAdCAM in Peyer's patches in the gut, VCAM and E-selectin in the skin and ICAM (1-3) in inflammatory areas.

There is further evidence that even at a late stage of functionality T cells that are largely responsible for the T cell help in aproinflammatory (THj) response express different adhesion molecules to the antibody response directed by TH2 cells or the uncommitted THC cells.

To do this, TH2 cells express CD40 ligand and bind to CD40 on the B cell. This triggers differentiation of these B cells into memory B cells producing Th2 associated antibody subclasses (IgG4 and IgE) whereas T helper cells that are polarized to THi proinflammatory cytokine production (IL-12, IL02, IFN-y) have different receptors for chemokines (CXCR3 and CCR5) to TH2 (IL-4, IL-13) producing cells (CCR3 and CCR4). This could be extremely relevant in the development of malignancy from these cells - for example Sezary/Mycosis fungoides CD4+ cells being the malignant counterpart of a TH2 type cell.

These complex homing mechanisms (rolling on endothelial cells, - integrin adhesion and chemoattraction at the destination site) allow selective trafficking to specific anatomical sites which in the case of T cells is particularly the skin and areas of chronic inflammation. This has great relevance to the T-cell lymphomas which are often found (or relapse in) T-cell anatomical areas particularly the skin, and it will be important to understand more about the homing behaviour of the normal lymphoid counterparts of malignant lymphoproliferative disorders as a common cause of death is due to extranodal spread , especially to the central nervous system.

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