Flow Cytometry of Hematological Malignancies

By Claudio Ortolani

Flow Cytometry of Hematological Malignancies contains an array of graphical outputs produced by the technique in the study of the most (and the least) common diseases. The images included allow you to compare your own results with a third party reference pattern.   There is a detailed description of the main leukocyte antigens, together with a description of their distribution amongst normal and abnormal blood cells. The book also provides a comprehensive description of the phenotype of every neoplastic blood disease recorded in the WHO classification system, including all the instructions needed to recognise and classify even the least common entity.

Designed to be practical, the book is perfect for quick consultation and is divided into two main sections. Section I deals with the direct object of immunophenotyping, and Section II deals with the ultimate target of the analysis. More than 50 antigens are covered and every antigen is dealt with in three main parts: general features, cytometric features and practical hints.

This authoritative and state-of-the-art reference will be invaluable for clinicians directly involved in the diagnosis and analysis of hematological diseases, including hematologists, hematopathologists, oncologists, pathologists and technicians working in diagnostic laboratories.

• Language: English
• Publisher: Wiley
• Release date: Jun 9, 2011
• ISBN: 9781444398052

Book preview

1 Antigens

CD1 Antigens

CD2 Antigen

CD3 Antigen

CD4 Antigen

CD5 Antigen

CD7 Antigen

CD8 Antigen

CD10 Antigen

CD11b Antigen

CD11c Antigen

CD13 Antigen

CD14 Antigen

CD15 Antigen

CD16 Antigen

CD19 Antigen

CD20 Antigen

CD22 Antigen

CD23 Antigen

CD24 Antigen

CD25 Antigen

CD30 Antigen

CD33 Antigen

CD34 Antigen

CD38 Antigen

CD45 Antigen

CD45 Isoforms

CD56 Antigen

CD57 Antigen

CD61 Antigen

CD64 Antigen

CD65 Antigen

CD66c Antigen

CD71 Antigen

CD79 Antigen

CD103 Antigen

CD117 Antigen

CD138 Antigen

HLA-DR Antigen

NG2 Antigen

Immunoglobulins

Myeloperoxidase

Cytotoxic Proteins

KIR, CD158 Isoforms

T Cell Receptor

Terminal Deoxy-nucleotidyl Transferase

Bcl-2 Protein

ZAP-70 Protein

References

CD1 Antigens

General features

CD1 antigens are a group of at least four different glycoproteins, named CD1a, CD1b, CD1c and CD1d, which weigh 43−49 kD. They are encoded by a group of genes situated on the long arm of chromosome 1 [1] and play a role in the presentation of lipidic and glycolipidic antigens to NKT cells [1,2].

CD1 antigens are mainly expressed on cells belonging to T and B lineages and on antigen presenting cells (APC).

As for the T lineage, CD1 antigens have been demonstrated on the membrane of the cortical or common thymocytes [3] and on the membrane of some T lymphocyte subsets in cord and neonatal peripheral blood [4]; a low expression of CD1 antigens can be demonstrated in the cytoplasm of T lymphocytes activated by phytohemagglutinin (PHA) in vitro [5].

As for the B lineage, both CD1c and CD1d have been demonstrated on the precursors and on some subsets of mature B lymphocytes. More precisely:

CD1c has been demonstrated on some subsets of B lymphocytes in the peripheral blood [6] [7], in the spleen [6,7] and in the mantle of the germinal center [6]

CD1c+ B lymphocytes account for the majority of B cells in tonsils [8], in cord blood [4], in the peripheral blood of newborns [4], and in the peripheral blood of subjects submitted to autologous or allogeneic bone marrow transplantation during the first year following transplant [7]

CD1d has been demonstrated on the membrane of bone marrow B precursors [9] and of B lymphocytes in peripheral blood [10], in the mantle of the germinal center [10], and in the spleen [11].

Finally, as for the antigen-presenting cells, CD1 antigens have been demonstrated on many cellular types. More precisely:

CD1a has been demonstrated on Langerhans cells [12], where it is expressed at an intensity of 1600 molecules per cell [13], on some CD11b+ CD14+ mononuclear cells reported in the peripheral blood of burnt subjects and interpreted as Langerhans cell precursors migrating from bone marrow to epidermis [14], on monocytes activated with GM-CSF in vitro [15], and on in vitro monocyte-derived dendritic cells [16]

CD1b has been demonstrated on monocytes activated with granulocyte macrophage-colony stimulating factor (GM-CSF) in vitro [15] and on a subset of Langerhans cells [17]

CD1c has been demonstrated on monocytes activated with GM-CSF in vitro [15], on Langerhans cells [17], and on a minor subset of myeloid dendritic cells characterized by CD11c++ CD123± phenotype [18]

CD1d has been demonstrated on resting monocytes [10], on dendritic cells of the dermis [19], and on in vitro monocyte-derived dendritic cells [19]

CD1a, CD1b, CD1c and CD1d have been demonstrated in the foam cells of the atherosclerotic plaque [20].

Cytometric features

The cytometric demonstration of molecules belonging to the CD1 family should be performed while taking the following points into account:

cytometric studies have demonstrated that activated T lymphocytes express CD1c on the membrane only when kept at room temperature, and fail to mount the molecule on the surface when kept at +4 or to +37°C [21]

the expression of CD1a on the surface of the leukemic blasts can fluctuate spontaneously after a short period of incubation in vitro [22].

The antibodies specific for CD1 antigens do not behave in the same way. It should be kept in mind that CD1a features four different epitopes, the first of which is recognized by clones D47, Na1/34 and L119, the second by clone L404, and the third by clone L504 [23]; it should be noted that CD1a on the cells of B cell chronic lymphocytic leukemia (B-CLL) can be demostrated only with clones other than OKT6 or Na1/34 [24].

The clones 7C4 and IOT6b recognize CD1b, and show different cellular reactivities as well [25].

Diagnostic features

CD1 antigens in neoplastic diseases of B cell precursors

CD1 antigens have been demonstrated in some cases of common acute lymphocytic leukemia [22]. In a group of 80 patients affected by childhood B lymphoblastic leukemia (B-ALL), the expression of CD1d has been demonstrated in 15% of the cases [9]. CD1d expression is significantly associated with pre-B phenotype, rearrangement of the gene MLL, and shorter global survival [9].

CD1 antigens in neoplastic diseases of T cell precursors

CD1 antigens are generally expressed on the cells of T lymphoblastic leukemia/lymphoma related to the stage of cortical or common thymocyte [3,22] (Fig. 1.1). According to the EGIL classification of T lymphoblastic leukemias, CD1 antigens are typically present in the T III form but missing in the T I, T II and T IV forms [26]. If CD13 is negative, the expression of CD1a is related to good survival [27], while the expression of CD1a together with CD10 is associated with the presence of the t(5;14) translocation [28].

Figure 1.1 Peripheral blood from a subject affected by T lymphoblastic leukemia (T-ALL). The blasts (red) express the phenotype CD45 dim+, CD1a+, CD3+ (heterogeneous), CD4+, CD8+.

CD1 antigens in acute myeloid leukemias

The expression of CD1a on the surface of the blasts of the acute myeloid leukemias has repeatedly been reported [22,29,30]. According to some authors, the expression of CD1a and CD1d is restricted to FAB subtypes characterized by a monocytic component [30].

CD1 antigens in neoplastic diseases of mature B cells

The presence of CD1 antigens on the surface of the elements of the neoplastic diseases of mature B cells has been demonstrated by different authors. More specifically, the cells of B cell chronic lymphocytic leukemia (B-CLL) have been reported to express CD1a [8,24], CD1c [6], and CD1d, whose intensity is more elevated in the cases without somatic hypermutations [31]; it is noteworthy that CD1a on B-CLL cells can be demostrated only with clones other than OKT6 or Na1/34 [24].

As for other neoplastic diseases of mature B cells, it has been reported that B cell prolymphocytic leukemia (B-PLL) cells express CD1c [8] [32], that Burkitt lymphoma (BL) cells do not express CD1c [6], that hairy cell leukemia (HCL) cells express CD1a [33] and CD1c [8], and that multiple myeloma (MM) cells express CD1d in the early stages, but tend to reduce its expression with disease progression [34].

CD1 antigens in neoplastic diseases of mature T and NK cells

The expression of CD1 antigens has sporadically been reported in rare cases of peripheral T cell lymphoma (PTCL) [35].

CD1 antigens in myelodysplastic and chronic myeloproliferative diseases

The expression of CD1d has been reported on the cells of the juvenile myelomonocytic leukemia (JMML) [30]. CD1a, CD1b and CD1c are expressed on the membrane of the blast cells in the 20% of cases of chronic myeloid leukemia (CML) in blastic crisis [22].

CD1 antigens in other pathological conditions

CD1a, CD1b and CD1c have been demonstrated with immunohistochemical techniques in the Langerhans cell histiocytosis (LCH) [36,37]. One of the most typical features of the pulmonary location of Langerhans cell histiocytosis (LCH) is the occurrence of more than 5% of CD1+ cells in the bronchoalveolar lavage (BAL) liquid [38].

CD1a expression has been reported in an anecdotal case interpreted as acute leukemia of Langerhans cell precursors on the basis of the presence of Birbeck granules and of the ability of blasts to develop dendritic processes when cultured in vitro [39].

CD1a has been demonstrated with immunohistochemical techniques in the indeterminate dendritic cell tumor (ICT) [40], but not in the follicular dendritic cell sarcoma (FDCS) nor in the interdigitating dendritic cell sarcoma (IDCS) [37].

CD2 Antigen

General features

CD2 is a 45−58 kD glycoprotein belonging to the superfamily of the immunoglobulins, which is encoded by a gene situated on the short arm of chromosome 1 [41]. CD2 is an adhesion molecule, constitutes the ligand of the CD58 molecule [41], and interacts with CD48 and CD59 molecules as well [42].

CD2 is normally expressed on thymocytes, on whose membrane it begins to appear at the prothymocyte level [43], and on mature T lymphocytes [44].

Not all mature T lymphocytes co-express CD2. Indeed, it is well known that in the peripheral blood, small subsets of T lymphocytes exist that show CD3+ CD2− phenotype, and are characterized by the expression of T cell receptor (TCR) either with alpha/beta [45] or gamma/delta chains [46].

The expression of CD2 is not restricted to the T lineage. Indeed, it is well known that CD2 is expressed:

on 70–90% of the NK cells negative for CD3 [47,48], where it is upregulated by activation [49]

on a minority of follicular dendritic cells (FDC) [50]

on a subset of mononuclear peripheral cells interpreted as precursors of myeloid dendritic cells [51]

on a subset of peripheral monocytes characterized by the co-expression of Fc epsilon receptor (FcepsilonRI) [52]

on a small subset of B cells in fetal liver [53], in fetal bone marrow [53], in thymus [54], in peripheral blood [55], and in the bone marrow of normal subjects [55].

Cytometric features

The staining of peripheral normal lymphocytes with an anti-CD2 monoclonal antibody generates a positive histogram with a narrow gaussian-like peak, clearly separated from the negative component, with a channel peak representing the presence of 24±7 E03 ABC (antibody binding capacity) [56].

Bimodal histograms can often be seen, especially when immune system activation is ongoing, because a higher number of CD2 molecules is expressed on activated cells [57] (Fig. 1.2).

Figure 1.2 The histogram produced by the cytometric analysis of CD2 is bimodal (A), because the activated CD25+ lymphocytes (red) express more CD2 molecules than CD25− lymphocytes (blue) (B).

In these cases the CD2 bright+ population tends to show higher values of forward and side scatter than the CD2 dim+ population.

CD2 bright+ cells nearly exclusively express CD45RO, while the CD2 dim+ population display either CD45RO or CD45RA [58]; the staining of CD2+ CD45RA+ cells with an anti-CD2 monoclonal antibody generates a histogram with a channel peak representing the presence of 21±4 E03 ABC while the staining of CD2+ CD45R0+ lymphocytes with the same MoAb generates a histogram with a channel peak representing the presence of 55±9 E03 ABC [56].

In accordance with the state of chronic activation caused by HIV infection, the lymphocytes of HIV-infected subjects seem to express a higher amount of CD2 molecules [59], while a reduced expression has been documented on the lymphocytes of elderly subjects [60]. According to some authors, the expression of CD2 on NK cells is dishomogeneous, being more intense in the CD16 dim+ CD56 bright+ subset than in the CD16 bright+ CD56 dim+ subset [61].

Not all the anti-CD2 MoAbs behave in the same way; some clones are able to inhibit the E-rosette formation [62], while others are able to activate T lymphocytes in vitro [63].

Diagnostic features

CD2 in neoplastic diseases of B cell precursors

Depending on the survey, the expression of CD2 has been reported in 1–4% of the observed cases [68,69,1065].

CD2 in neoplastic diseases of T cell precursors

CD2 is generally expressed on the blasts of the neoplastic diseases of T cell precursors, but it may be missing in the most immature forms [64]. According to the EGIL classification of T lymphoblastic leukemias, CD2 is typically present in the T II, T III and T IV forms, but is missing in the most immature form, T I [26]. CD2 is generally expressed by the cases with TCR alpha/beta, but only by some cases with TCR gamma/delta [65,66]; its presence in childhood cases is correlated with an increased probability of maintaining complete remission [67].

CD2 in acute myeloid leukemias (AML)

Depending on the survey, the expression of CD2 has been reported in 3–34% of the observed cases [70−77]. CD2 seems to be frequently expressed:

on the blasts of pediatric AML-M2 negative for translocation t(8;21) [78]

on the promyelocytes of AML-M3, with particular predilection for the microgranular variant (AML-M3v) [76,79,80], and for the presence of the short type of the PML-RARA fusion gene [77,80]

on both the monocytic and non-monocytic neoplastic cells of AML-M4 [76,81,82]

on the blasts of AML-M5 [82].

The presence of CD2 (and also of CD4, CD7 and CD56) on the blasts of AML is correlated with an increased risk of extramedullary disease (granulocytic sarcoma, and cutaneous, gingival and meningeal involvement) [83], and with a lower incidence of complete remission [84]. The CD2 expression has been reported in cases of AML with morphological anomalies mimicking the picture of Chediak–Higashi disease (pseudo Chediak–Higashi, PCH) [85], and in some cases of blastic plasmacytoid dendritic cell (BPDC) neoplasm [86]. The presence of CD2 on AML-M3 promyelocytes correlates with the occurrence of thrombotic events [87].

In AML-M4 with inv(16)/t(16;16), the expression of CD2 is variable, and has been reported as weaker in cases with fusion transcript CBFbeta-MYH11 other than type A [1736].

CD2 in neoplastic diseases of mature B cells

Sporadic reports exist signaling the presence of CD2 in isolated cases of B lineage non-Hodgkin lymphoma [88,89]. Since CD2 has been demonstrated on the surface of normal B lymphocytes [55], it is theoretically possible that these cases constitute a clonal expansion of very infrequent normal B cells rather than an expansion of B cell with an aberrant phenotype.

The expression of CD2 has occasionally been demonstrated in the sporadic B cell chronic lymphocytic leukemia (B-CLL) [55], but it seems particularly frequent in familial B-CLL, where it appears in 13% of the cases [90]; the demonstration of CD2 on the cells of a patient affected by B-CLL suggests that clinical investigations should be extended to the relatives as well [90].

Furthermore, CD2 has been demonstrated in some cases of follicular lymphoma (FL) [55], in some cases of diffuse large B cell lymphoma (DLBCL) [55,91], in some cases of diffuse large B cell lymphoma associated with pyothorax (PAL) [92], in some cases of hairy cell leukemia (HCL) [55], and in a case of multiple myeloma (MM) [93].

CD2 in neoplastic diseases of mature T and NK cells

CD2 is generally expressed on the cells of the neoplastic diseases of mature T and NK cells, but it may also be missing or expressed in an aberrant way. In the peripheral T lymphoma not otherwise specified (PTCLnos), about a third of the cases has been reported to show an aberrant antigen expression [94,95]. An aberrant CD2 expression has been reported with immunohistochemical methods in atypical cutaneous T cell infiltrates of subjects affected by mycosis fungoides [96], and with flow cytometric methods on neoplastic lymphocytes of subjects affected by Sézary syndrome [97], by T cell chronic lymphocytic leukemia (T-CLL) and by adult T cell leukemia/lymphoma (ATLL) [98].

The CD2 expression is more constant in the cases of angio-immunoblastic T cell lymphoma (AITL) [95], while in T cell large granular lymphocytic leukemia (T-LGL) it has been reported either as constant [99] or as variable [100]. The cases of CD8+ cutaneous T cell lymphoma (CD8+ CTCL) with CD2+ CD7− phenotype show a better prognosis than those with phenotype CD2− CD7+ [101].

In the neoplastic diseases of mature NK cells, CD2 may be missing [102] but it has been reported in most cases of chronic NK cell lymphocytosis (CNKL) [102–104], of aggressive NK cell leukemia (ANKL) [105,106], and of NK lymphoma [107].

CD2 in myelodysplastic and chronic myeloproliferative diseases

CD2, which is usually missing on normal mast cells [108], has been reported together with CD22 and CD25 on the neoplastic mast cells in systemic mastocytosis and mast cell leukemia [109–111]. Moreover, CD2 has been reported in a third of cases of chronic myelomonocytic leukemia (CMML) [82].

CD2 in other pathological conditions

CD2 has been demonstrated by immunohistochemical methods on the membrane of the cells of Langerhans cell histiocytosis (LCH)[36].

CD3 Antigen

General features

CD3 is made up of five different chains, i.e. gamma, delta, epsilon, zeta and eta. Chains gamma, delta, epsilon, and eta are encoded by a gene on the long arm of chromosome 11 [112], while chain zeta, separately clustered as CD247 [113], is encoded by another gene on the long arm of chromosome 1 [114]. In T cells, CD3 transmits the activation signal produced by the engagement of TCR [115,116].

Stechiometrical ratios between CD3/TCR components have not yet been completely understood. There is evidence that the CD3/TCR complex forms a multimeric array together with the tyrosine-phosphatase CD45, with a tyrosine-kinase, and with the CD7, which takes part in signal transmission [117].

The expression of the delta and epsilon chains is restricted to T lymphocytes, with two important exceptions:

the fetal and adult activated NK cells, which can contain delta and epsilon chains in the cytoplasm [118,119]

the plasmacytoid dendritic cells, in the cytoplasm of which epsilon chains have been demonstrated [120].

An isolated report exists, according to which eosinophils express low levels of CD3 together with a functional gamma/delta TCR [121].

As a rule, gamma and zeta chains are present in NK cells as well [122], as either homodimers or heterodimers [123]. In NK cells, both chains are not covalently linked with the transmembrane tail of the CD16, and transmit the signal produced by the linkage between CD16 and the IgG crystalizable fragment [124–126]. Signal transduction by zeta chain is carried out by the intracytoplasmic protein ZAP-70 [127,128].

During normal T cell maturation, the CD3 appears in the cytoplasm at the prothymocyte level, but is only expressed on the membrane from the common thymocyte stage on [3,129–134].

During normal T cell maturation, the TCR and the CD3 complex are assembled together before they are expressed on the surface [135]; consequently, the TCR is not normally expressed on the membrane in the absence of CD3, and vice versa [136,137].

CD3 has been demonstrated with immunohistochemical methods in the cytoplasm of Warthin–Finkeldey polykaryocytes, which can be seen in tonsils during the measles prodromic period, and are probably derived from T lymphocytes [138].

Cytometric features

Almost all anti-CD3 monoclonal antibodies are specific for an epsilon chain epitope [139,140]. The staining of peripheral normal lymphocytes with an anti-CD3 epsilon monoclonal antibody generates a positive histogram with a narrow gaussian-like peak, clearly separated from the negative component, with a channel peak representing the presence of 57±7 E03 ABC [56].

Evidence does exist that the number of CD3 epsilon chains is not the same for every T lymphocyte, but is particularly high on gamma/delta T cells [141], which express roughly 116±15 E3 ABC per cell [142].

Among peripheral T lymphocytes, CD3 expression tends to vary depending on the T lymphocyte subset. Evidence does exist that, in comparison to CD8 bright+ T lymphocytes, the CD3 mean fluorescence intensity (MFI) of positive cells is almost twice as intense in CD4+ T cells, while T CD8 dim+ lymphocytes behave similarly to CD4+ lymphocytes. This behavior does not depend on cellular dimensions, inasmuch as in CD4+ lymphocytes scatter values are even lower than in CD8 bright+ ones [58].

A reduced expression of CD3 has been reported in other cases:

in alveolar T cells, with a negative modulation greater for CD4+ cells [143]

in activated T cells that infiltrate nasal polyps [144]

in intrathyroidal T lymphocyte subsets in autoimmune thyroid disease [145]

in intestinal intraepithelial T lymphocytes [146]

in T cells of patients given OKT3 rescue treatment for kidney rejection [147]

in T cells of patients with HIV infection [59,148]

in T cells of aged subjects [60]

in a minor subset of peripheral T lymphocytes characterized by low CD4 expression, and positivity for CD25 and HLA-DR [149].

This CD3 downmodulation might be due to the activation state common to the great majority of the cases reported; it is important to bear in mind that a CD3 downmodulation can be caused by apoptosis as well [150].

In some cases, the positive histogram can appear with a bimodal shape, mostly due to the presence of a consistent subset of gamma/delta T cells, which actually bear more TCR/CD3 epsilon complexes than alpha/beta T cells on the membrane [142] (Fig. 1.3).

Figure 1.3 Pattern of expression of T-specific CD3 antigen on peripheral T lymphocytes. The positive peak can show a bimodal appearance (A-C), because gamma/delta T lymphocytes (red) express more CD3 molecules than alpha/beta T lymphocytes (B,D).

In our experience, this behavior does not occur with the gamma/delta T lymphocytes mounting Vdelta1/Jdelta1 sequences stained by deltaTCS1 MoAb (Fig. 1.4).

Figure 1.4 Different patterns of expression of T-specific CD3 antigen on peripheral gamma/delta T lymphocytes. Vdelta1/Jdelta1+ gamma/delta T lymphocytes (red) express CD3 less brightly than Vdelta1/Jdelta1− gamma/delta T lymphocytes (blue). Vdelta1/Jdelta1+ lymphocytes were recognized by MoAb delta-TCS-1, and TCR gamma/delta lymphocytes were recognized by MoAb 11F2.

Sometimes it is possible that the bimodality of the CD3+ peak is due to the presence of a clonal T cell population, homogeneously expressing the molecule at an intensity that differs from other normal residual T cells. This behavior is frequently reported in patients affected by mature T cell malignancies [94,151].

In comparison to mature T cells, thymocytes express CD3 with a different intensity; as a rule, most common or cortical CD1+ CD4+ CD8+ thymocytes express low amounts of CD3, while mature or medullar CD1− and CD4+ or CD8+ thymocytes express the molecule in the same way as mature T lymphocytes [130,152], with a differential higher expression on CD4+ CD8− T cells [58].

A thymocyte CD4+ CD8+ subset has been reported expressing high levels of CD3; it is hypothesized that this subset is a late differentiation stage between cortical and medullar thymocytes [153].

As mentioned previously, the CD3 can be looked for both on the membrane and in the cytoplasm of the cell. The demonstration of the intracytoplasmic molecule requires the use of permeabilization techniques which allow intracellular entry of the antibody. Although they could be improved by some optimization procedures [154], such techniques can rely on the use of standardized commercial permeabilizing solutions [155–158].

MoAb OKT3, SK7/Leu4 and UCHT-1

The three monoclonal antibodies OKT-3, SK7/Leu4 and UCHT-1 recognize CD3 epsilon chains in cells transfected with genes coding for epsilon and delta chains or for epsilon and gamma chains, but do not recognize CD3 epsilon chains in cells transfected with genes coding for epsilon chains only [159]. This behavior suggests that the three antibodies recognize a conformational epsilon chain epitope, depending on the association of epsilon chain with delta or gamma chain, and are not able to detect isolated intracytoplasmic epsilon chains [159].

Consequently, a negativity for intracytoplasmic epsilon chains accomplished with one of the aforementioned antibodies is not sufficient proof of epsilon chain absence, and should be validated using an antibody specific for isolated epsilon chains, such as SP34 and APA 1/1, or a polyclonal rabbit antiserum raised against a synthetic polypeptide mimicking a sequence on the intracytoplasmic tail of the epsilon chain [160].

This point is of some practical importance. Given that in thymocyte cytoplasm delta and epsilon chains are simultaneously expressed from the prothymocyte level onwards [161], these three antibodies are perfectly suitable for demonstrating the intracytoplasmic CD3 antigen in T cell malignancies, but could miss it in some cases of NK neoplasms. It has been reported that MoAb UCHT-1 is able to stain the cerebellar Purkinje cells [162].

MoAb WT31

In the same way as OKT-3, SK7/Leu4 and UCHT-1, the monoclonal antibody WT31 recognizes CD3 epsilon chains in cells transfected with genes coding for epsilon and delta chains or for epsilon and gamma chains, but do not recognize CD3 epsilon chains in cells transfected with genes coding for epsilon chains only [159]. This behavior confirms that, contrary to the original hypothesis [163] and in keeping with successive remarks [139], MoAb WT31 is not specific for a TCR alpha/beta determinant, but binds a conformational epitope on CD3 epsilon chains, and should be considered a bona fide anti-CD3 antibody.

Nevertheless, it should be stressed that the epitope stained by MoAb WT31 is particularly accessible to this MoAb in the case of TCR alpha/beta co-expression; this condition makes MoAb WT31 fit for the presumptive identification of TCR alpha/beta T cells, especially if used in combination with a second antibody specific for the same chain. In this case, the sterical hindrance between the two antibodies blocks the binding between WT31 and the epsilon chain of T cells bearing gamma/delta TCR, and WT31 behaves like a MoAb specific for alpha/beta TCR only.

In these conditions, the staining of peripheral normal T lymphocytes with the WT31 monoclonal antibody generates a histogram with a negative peak encompassing T cells bearing gamma/delta TCR (Fig. 1.5).

Figure 1.5 If lymphocytes are stained with an anti-CD3epsilon antibody (MoAb SK7 in the reported example), MoAb WT31 does not recognize gamma/delta T lymphocytes (red), and behaves like a bona fide anti-TCR alpha/beta MoAb.

The removal of the sterical hindrance allows the WT31 monoclonal antibody to bind the epsilon chain of T cells with gamma/delta TCR, although in a weaker way than alpha/beta T cells. Indeed, if we stain a sample containing a high number of gamma/delta T cells using both the WT31 monoclonal antibody and a second monoclonal antibody specific for TCR gamma/delta, the WT31 monoclonal antibody will generate a histogram with a first positive peak which encompasses gamma/delta negative T cells, and a second positive but intermediate peak which encompasses gamma/delta positive ones (Fig. 1.6).

Figure 1.6 Without the steric hindrance caused by the anti-CD3epsilon antibody, Moab WT31 recognizes gamma/delta T lymphocytes (red) as well, although more weakly than alpha/beta ones.

From a practical point of view, the possibility of sterical hindrance between the WT31 MoAb and another anti-CD3 epsilon monoclonal antibody suggests that a sequential staining procedure should be performed, in which the sample is incubated first with WT31 alone and then with the other anti-CD3 epsilon antibody.

MoAb T3

The FITC-conjugated form of T3 displays unexpected behavior [164]. In a multicolor analysis which combines a MoAb specific for TCR gamma/delta (clone 11F2) and a second anti-CD3 epsilon MoAb (clone SK7), the FITC-conjugated form of T3 does not recognize gamma/delta T cells (Fig. 1.7).

Figure 1.7 When conjugated with FITC, MoAb T3 does not recognize gamma/delta T lymphocytes (red).

It is interesting to notice that in this model, T3-FITC behaves very similarly to WT31, which is shown for comparison (Fig. 1.8).

Figure 1.8 Comparison between WT31 and T3-FITC monoclonal antibodies. Neither recognizes gamma/delta T lymphocytes (red).

The anomalous behavior of T3-FITC is difficult to explain. The small molecular volume of FITC rules out a sterical hindrance effect, and the independence of the phenomenon from the length of incubation does not suggest affinity variations induced by the conjugation procedures.

It has been observed that, owing to a different glycosylation pattern, CD3 delta chains in gamma/delta T cells display a more acidic isoionic point than CD3 delta chains in alpha/beta T cells [165]. It could be hypothesized perhaps that FITC increases the total negative charge of the FITC-conjugated antibody, allowing its binding with CD3 delta chains in alpha/beta T cells, but preventing its binding with the more glycosylated CD3 delta chains in gamma/delta T cells.

Other antibodies

Some antibodies do exist that are able to recognize isolated CD3 epsilon chains. These include the monoclonal antibody SP-34 [159], APA 1/1 [159] and F7.2.38 [166], as well as a polyclonal rabbit antiserum [160] which displays a high cross-specificity and is even able to react with Australian koala’s T lymphocytes [167]. Monoclonal antibody SP-34 can recognize isolated epsilon chain either on the membrane or in the cytoplasm [159], and is able to identify T cells from all but two non-human primate species tested and from the Siberian tiger as well [168].

Clones F7.2.38 and APA 1/1 have been raised against an intracellular epitope, and can consequently recognize only intracellular chains [166]. Either the rabbit polyclonal antibody or the monoclonal antibody F7.2.38 reacts with isolated CD3 epsilon chains in formalin-fixed paraffin-embedded tissue samples. This is a very important point, because their use in immunohistochemistry allows the detection of intracellular isolated CD3 epsilon chains in NK lymphoma cells [169–171].

Other interesting clones are clone F101.01, which displays a behavior similar to clone WT31 [172], and clone 446, which cross-reacts with a determinant in the cytoplasm of basal keratinocytes [173].

Furthermore, several antibodies anti-CD3 zeta chains are commercially available, among which clone TIA-2 should be cited [174]. This antibody reacts with an intracytoplasmic epitope, and requires the permeabilization of the sample [175].

Diagnostic features

It is important to bear in mind that in acute leukemia characterization as well as in every doubtful case, the CD3 antigen must be looked for both on the surface and in the cytoplasm [176] (Fig. 1.9).

Figure 1.9 Combined membrane and cytoplasmic CD3 staining in a case of T lymphoblastic leukemia. The technique allows the distinction between mCD3+/cyCD3+ residual lymphocytes (blue) and mCD3−/cyCD3+ leukemic blasts (red).

Nevertheless, it should be stressed that the presence of intracytoplasmic CD3 epsilon chains is not necessarily to be interpreted as proof of T lineage attribution, given that CD3 epsilon chains can be demonstrated in NK cell lines [118,177], in NK malignancies [171,177,178], in some cases of the so-called myeloid/NK cell precursor acute leukemia [179], in plasmacytoid dendritic cells [120], and in some cases of plasmacytoid dendritic cell-derived neoplasms [180,181].

CD3 in neoplastic diseases of B cell precursors

As a rule, CD3 is not detected in neoplastic diseases of B cell precursors [69,182,183]. Nevertheless, cytoplasmic CD3 has been detected in the blasts of two isolated cases of B lymphoblastic leukemia (B-ALL), one of which featured the combined expression of CD2 and CD19 [184]. Owing to the high likelihood of technical artefacts in intracellular antigen detection, the greatest care must be taken in the interpretation of these data.

CD3 in neoplastic diseases of T cell precursors

CD3 is usually detectable in the cytoplasm of every case of neoplastic disease arising from T cell precursors. According to the EGIL classification of T lymphoblastic leukemias, CD3 is detectable on the membrane of the cases pertaining to the most mature form, T IV, but is lacking on the membrane of the cases pertaining to the more immature forms T I, T II, and T III [26,185].

Actually, this classification is a little rigid, and it is possible to find cases with cells co-expressing CD1 and CD3 on the membrane. The absence of the CD3 surface antigen correlates with the expression of CD13, CD33, CD34 and CD56 [186].

The CD3 surface antigen can be demonstrated in a proportion of cases varying from 30% to 40% of total cases [186−187]; in childhood leukemias, the presence of the CD3 surface antigen seems to confer an increased risk of treatment failure [189].

However, the CD3 surface antigen is expressed by T lymphoblastic leukemia (T-ALL) blasts more faintly than by mature residual T cells [58], and this difference can be used in the cytometric determination of the minimal residual disease.

Some cases of lymphoblastic lymphoma have been reported, categorized as NK precursor lymphomas because of the CD16 expression, in which the CD3 has been demonstrated in cytoplasm but not on membrane [190−192]. One case of acute lymphoblastic leukemia has been reported, categorized as T/NK precursor leukemia, whose cells displayed a mCD3-/cyCD3+ phenotype and expressed the following antigens: CD1a, CD2, CD4, CD13, CD19, CD30, CD33 and CD56 [193].

CD3 in acute myeloid leukemias

CD3 has been sporadically detected either on membrane or in cytoplasm of AML cells [29,76,194−198]. More specifically, CD3 intracytoplasmic antigen has been detected in two out of 13 cases of AML-M3v [199].

The phenotype mCD3-/cyCD3+ has been detected in the so-called acute leukemia of myeloid/NK precursors [200,201], a clinical entity not recognized by the 2008 WHO classification [202] (Fig. 1.10). This phenotype has also been detected in the cells of some cases of blastic plasmacytoid dendritic cell (BPDC) neoplasm [180,181].

Figure 1.10 A putative case of M/NK-AL, whose blasts (red) display a typical phenotype (CD7+, CD56+, CD34+) (C,D) and weak positivity for cytoplasmic CD3 (E,F).

The hypothesis can be put forward that at least some of the AML CD3+ cases are the consequence of the neoplastic transformation of a T/myeloid precursor able to retain the phenotypic features of both the evolutionary lineages. With regard to this point, it is interesting to stress that 14 cases of AML have been reported in which the consensual presence of cyCD3, MPO, CD2 and CD7 could be demonstrated. These cases displayed special features such as high blast count, presence of lymphadenopathies, poor response to induction protocols specific to myeloblastic leukemias, and good response to induction protocols specific for lymphoblastic leukemias [203,204]. These cases, provisionally defined as acute myeloid leukemia with T-lymphoid features [203,204], seem very similar to another six cases, categorized as mixed lineage leukemias, whose blasts displayed the following phenotype: CD1−, CD4−, CD8−, CD7+, MPO+, cyCD3+ [205,206].

CD3 in neoplastic diseases of mature B cells

As a rule, CD3 expression is not detected in neoplastic diseases of mature B cells [207]. However, some positive anecdotal cases have been reported:

one B cell chronic lymphocytic leukemia (B-CLL) case characterized by the presence of translocation t(18;22), whose cells expressed CD3 and CD8 beside the normally expected B cell markers [208]

four cases of otherwise typical hairy cell leukemia (HCL) whose cells reacted with UCHT-1 but not with OKT-3 MoAb [209]

one case of primary effusion lymphoma (PEL) whose cells were positive for intracytoplasmic CD3 [210]

a little casistic made up of one B cell non-Hodgkin lymphoma (B-NHL) and six B-CLL cases, whose cells co-expressed CD2 and CD3, and displayed IgH but not TCR gene rearrangement [88]

one case of multiple myeloma (MM) [93]

four cases of diffuse large B cell lymphoma (DLBCL) [91].

CD3 in neoplastic diseases of mature T and NK cells

The cells of the neoplastic diseases of mature T cells are usually but not always positive for CD3 (Fig. 1.11). An immunohistochemical study detected the presence of CD3 in no more than 71% of the cases of peripheral T cell lymphomas, not otherwise specified (PTCLnos), in no more than 60% of the cases of angio-immunoblastic T cell lymphoma (AITL), and in no more than 26% of the cases of systemic anaplastic large cell lymphoma (ALCL), with a significantly more frequent expression in the ALK1-positive cases [211]. Another independent study has confirmed the particularly low frequency of CD3 positivity in ALCL and other CD30+ T chronic lymphoproliferative diseases [1738].

Figure 1.11 Aberrant CD3 expression on neoplastic CD4+ cells (red) in four cases of neoplastic disease of mature T cells. (A) angioimmunoblastic T cell lymphoma (AITL), (B) anaplastic large cell lymphoma (ALCL), (C) T cell prolymphocytic leukemia (T-PLL), (D) peripheral T cell lymphoma, not otherwise specified (PTCLnos).

Sometimes the CD3 can be expressed in an aberrant way, i.e. with less or more intensity than in normal mature T cells, and sometimes it may be detected in the cytoplasm, but not on the cell surface [94].

In peripheral T cell lymphoma not otherwise specified (PTCLnos), CD3 is aberrantly expressed in a proportion of cases varying from 6% to 66% of the total [35,94,212–220]. In the more recent studies, the higher percentage of aberrations is probably due to the increasing sensitivity of modern cytometric techniques.

An aberrant CD3 expression has been reported in T prolymphocytic leukemia (T-PLL) [221,222], angioimmunoblastic T cell lymphoma (AITL) [223,224], T cell large granular lymphocytic leukemia (T-LGL) [225,226], Sézary syndrome (SS) and mycosis fungoides (MF) [151,227–230], adult T cell leukemia/lymphoma (ATLL) [231–235], and enteropathy-associated T cell lymphoma (EATCL) [236]. According to a recent report, the gamma/delta T cells of hepatosplenic T cell lymphoma (HSTCL) typically express CD3/TCR complex at a lesser intensity than normal gamma/delta T cells [237].

Neoplastic populations of T cells with mCD3-/cyCD3+ phenotype have been detected in the peripheral blood in patients affected by angioimmunoblastic T cell lymphoma (AITL) [238] and in a subset of patients affected by hypereosinophilic syndrome. In this last case, hypereosinophilia represents a paraneoplastic response to the overproduction of IL-5 by the pathological T clone [239].

The cells of the neoplastic diseases of mature NK cells do not express the CD3/TCR complex on the membrane, but can contain free CD3 epsilon and CD3 delta chains in the cytoplasm. Accordingly, the phenotype mCD3-/cyCD3+ is a typical although not exclusive feature of this type of disease, and it has been documented in NK lymphoma in either nasal [107,169,171,177,178,240–246] or nasal-type [107,177,241,243,244, 247–249], and in aggressive NK leukemia (ANKL) [105,244,250].

CD3 in myelodysplastic and chronic myeloproliferative diseases

The bone marrow of patients affected by refractory anemia with blast excess can host a consistent percentage of cells characterized by the expression of lymphoid antigens CD3 and CD7 along with the expression of myeloid antigens CD13 and CD33. This feature has been explained by the neoplastic transformation of a pluripotent precursor able to retain the phenotypic features of both lineages [251].

Finally, CD3 has also been detected on the blasts of transient abnormal myelopoiesis (TAM), also known as transient myeloproliferative disorder (TMPD), occurring in newborns affected by Down syndrome [252].

CD3 in other pathological conditions

In some cases of so-called myeloid and lymphoid neoplasm with FGFR1 abnormalities, also known as 8p11 stem cell syndrome, the expression of CD3 has been demonstrated by immunohistochemistry not only in T lymphoblasts but in myeloid blasts as well [253].

CD4 Antigen

General features

CD4 is a 55 kD glycoprotein belonging to the superfamily of the immunoglobulins and encoded by a gene situated on chromosome 12 [41,254]. CD4 is a transmembrane molecule constituted by four extracellular domains similar to those of immunoglobulins [255]. Amino-terminal domain 1 contains an epitope acting as receptor for the gp120 of HIV-1 virus [256], while domains 1 and 2 act together as receptor for the HLA-DR antigen [257], binding its non-polymorphic domain beta2 [258]. CD4 acts as a receptor for IL-16 as well [259].

CD4 is mainly expressed by a subset of T lymphocytes with TCR alpha/beta, on whose membrane it begins to appear at the thymocyte level. In particular, CD4 appears first on immature thymocytes lacking CD8. These single positive immature thymocytes are characterized by the CD1a+, CD4+, CD8− phenotype. In the course of their maturation, these elements begin to co-express CD8, giving origin to the CD1a+ CD4+ CD8+ double-positive thymocytes, and then finally segregate into two populations of single positive mature thymocytes, which express the mutually exclusive phenotypes CD4+ CD8− and CD4− CD8+ [130,1742].

Unlike T lymphocytes with TCR alpha/beta, T lymphocytes with TCR gamma/delta do not express CD4. Nevertheless, it has been reported that at least in some subjects, gamma/delta T lymphocytes express CD4 at a frequency ranging between 1% and 4% [260]; this frequency can be increased in vitro by infection with HHV-6 virus [261]. CD4 is also expressed by immature NKT cells [2], by a subset of mature NKT cells [2], and by antigen presenting cells (APC), including monocytes [262], macrophages [262], myeloid dendritic cells [263], plasmacytoid dendritic cells [263], Langerhans cells [262], activated microglial cells [264], dendritic cells of cord blood [265] and follicular dendritic cells (FDC) [50], that seem selectively not to express the epitope recognized by the clone OKT4D [50].

CD4 is expressed on hemopoietic [266] and erythroid precursors [267], and it has been reported on a subset of neutrophils in some subjects who were either healthy or affected by HIV infection [268]. CD4 can be induced on basophils [269] and it is constitutively although weakly expressed on eosinophils [270], but not on mast cells [109]. Finally, CD4 has been documented on tonsillar activated B cells [271], on a minority of NK cells [272], and on activated CD8 bright+ T lymphocytes in the course of HIV infection [273].

Cytometric features

The monoclonal antibodies OKT4, OKT4A, OKT4B, OKT4C and OKT4D recognize different epitopes [274], and it is known that some clones, such as Leu3a, OKT4A and F101–69, recognize epitopes related to the binding site for the gp120 protein of HIV-1 virus [275].

In the human, the distribution of CD4 epitopes depends on a genetically determined polymorphism, consisting of the substitution of a molecule of tryptophan for a molecule of arginine in position 240 [276]. Subjects carrying this polymorphism do not express the epitope recognized by the MoAb OKT4, but normally express the epitopes recognized by OKT4A and Leu3a MoAbs. In these subjects, OKT4 MoAb does not recognize CD4, or produces positive histograms with a peak channel consistent with an intensity of expression about half that of normal controls [277]. This anomaly is exceptionally reported in Caucasians [278], is rare (<1%) in the Japanese population [277], but is relatively frequent in populations of African origin [279]. It must finally be remembered that the phenotype OKT4–/Leu3a+ is not the only possible anomalous phenotype; an isolated report exists about an apparently normal subject selectively lacking the epitope recognized by Leu3a MoAb [280].

The staining of peripheral normal lymphocytes with an anti-CD4 MoAb generates a positive histogram with a narrow gaussian-like peak, clearly separated from the negative component, with a channel peak representing the presence of 50±10 E03 ABC [56], equivalent to the presence of around 100 E03 molecules of CD4 per cell [281].

Besides this population, operationally defined CD4 bright+, there is sometimes a second little population of CD4+ lymphocytes characterized by a reduced expression of the antigen (about 50 E03 molecules per cell). These CD4 dim+ lymphocytes account for 5–10% of all the peripheral lymphocytes, are characterized by a reduced expression of CD3, co-express CD25 and HLA-DR, are increased in old age, and have been interpreted as chronically activated and apoptosis-resistant lymphocytes [60,149] (Fig. 1.12).

Figure 1.12 Differential CD4 expression on T lymphocytes in a subject affected by HIV infection. CD3+ CD4 dim+ lymphocytes (A, red) tend to display a CD45RA−, CD62L+ phenotype (B).

A reduced expression of CD4 on T lymphocytes has been documented in subjects affected by B-CLL [282] and by Nijmegen breakage syndrome (NBS) [283].

Finally, it should be borne in mind that thymocytes and monocytes express the antigen at a lesser extent than mature T lymphocytes [284]; in particular, CD4 is expressed on peripheral monocytes with an intensity of 17±5 E0 ABC [56] (Fig. 1.13). Such intensity is further reduced on the monocytes of newborns [285] and traumatized patients [286].

Figure 1.13 CD4 antigen is expressed by monocytes (red) at a lower intensity than by T lymphocytes (blue).

Diagnostic features

CD4 in neoplastic diseases of B cell precursors

As a rule, the expression of CD4 is missing in the neoplastic diseases of B cell precursors, but it has been reported in some isolated cases [69,1065].

CD4 in neoplastic diseases of T cell precursors

On the blasts of the neoplastic diseases of T cell precursors, CD4 can be expressed alone or together with CD8 on the forms derived from the common thymocytes.

The isolated expression of CD4 suggests a disease stemming from an immature single-positive precursor characterized by a CD1a+ CD4+ CD8− phenotype and situated immediately before the stage of double -positive CD4+ CD8+ common thymocyte.

In a group of cases made up of 18 patients affected by T lymphoblastic leukemia (T-ALL), the single-positive CD4+ CD8− phenotype has been reported in 39% of cases, while the double-positive CD4+ CD8+ phenotype has been reported in 22% [287]. Contrary to what happens in the neoplastic diseases of mature T cells, in the neoplastic diseases of T cell precursors the presence of the TCR gamma/delta does not rule out CD4 expression [288].

CD4 in acute myeloid leukemias

Depending on the survey, the expression of CD4 has been reported in 36–74% of the observed cases [71,73–75,77,289].

According to some authors, CD4 expression is strongly indicative of a myeloid lineage [290] independently of a monocytic commitment [291], but for others CD4 expression is frequent in the AML-M4 and AML-M5 forms [29,77], with a preference for the most mature cases [73]. In a survey made up of 495 adult patients, CD4 expression has been documented in 45.9% of the cases, and in 37.4% of AML-M1, 33.7% of AML-M2, 35.4% of AML-M3, 65% of AML-M4, 78.3% of AML-M5, and 55.6% of AML-M6 [289]. According to some authors, the absence of CD4 characterizes the pediatric AML-M2 with translocation t(8;21) [71], while other authors report that in a survey of 59 pediatric cases, CD4 was regularly co-expressed in all the six observed cases [77].

It is interesting to note that in the study of Abdelhaleem and co-workers, CD4 was co-expressed in all the AML-M7 cases [77]; the expression of CD4 in this FAB subtype has been reported in another case [289]. In an isolated case of monoblastic leukemia, CD4 was the only antigen found positive in the absence of myeloid and monocytic lineage-specific markers [292].

The presence of CD4 on the blasts of AML correlates to an increased risk of extramedullary disease (granulocytic sarcoma, and cutaneous, gingival and meningeal involvement) [83], to anomalies of chromosome 11 [289], and to the pericentric inversion of chromosome 16 [289].

CD4 has been demonstrated with immunohistochemical techniques in an isolated case of myeloid sarcoma [293], and in some cases of the so-called acute leukemia of myeloid/NK precursors (M/NK-AL) [294], a clinical entity not recognized by the 2008 WHO classification [202].

CD4 expression, together with the expression of CD56 and CD123 and the absence of other lineage-specific markers, constitutes the characteristic phenotype of the blastic plasmacytoid dendritic cell (BPDC) neoplasm [86,295–299].

CD4 in neoplastic diseases of mature B cells

CD4 expression is usually missing in neoplastic diseases of mature B cells, but it has been reported with immunohistochemical techniques in most of the cases belonging to a rare variety of diffuse large B cell lymphoma (DLBCL), named ALK-positive large B cell lymphoma (ALK+ LBCL) [300,301].

CD4 expression has also been reported in sporadic cases of a not well-defined DLBCL with primary splenic onset [302], DLBCL associated with pyothorax (PAL) [92], hairy cell leukemia (HCL) [303], multiple myeloma (MM) [93,304], plasma-blastic lymphoma (PBL) [91,304], and B cell chronic lymphocytic leukemia (B-CLL) [305], and in an isolated case of Burkitt lymphoma with plasmacytoid differentiation arising in a subject with HIV infection [1731].

CD4 in neoplastic diseases of mature T and NK cells

In the neoplastic diseases of mature T cells with leukemic presentation, CD4 is generally expressed:

in most cases of T cell prolymphocytic leukemia [306]

in most cases of adult T cell leukemia/lymphoma [307–309]

in most cases of Sézary syndrome (SS) [97,310–312] and mycosis fungoides (MF) [313]

in a minority of cases of T cell large granular lymphocytic leukemia (T-LGL) [314–317].

As for the nodal lymphomas, CD4 is expressed on the cells of a percentage of cases of angioimmunoblastic T cell lymphoma (AITL) ranging from 40% to the virtual totality of cases [95,318–320], and on the cells of most cases of anaplastic large cell lymphoma (ALCL) [321,322], with particular predilection for the ALK-positive cases [323].

The elements of peripheral T cell lymphoma not otherwise specified (PTCLnos) express CD4 in most cases, with percentages ranging between 40% and the virtual totality of cases, depending on the survey [35,94,95,324,325]; a subtype of peripheral T cell lymphoma (PTCL) exists, which is characterized by predominant involvement of lymphoid follicles, translocation t(5;9), and constant expression of CD3+, CD5+, CD4+, and bcl-6+ phenotype [326]. As for the other morphological variants of PTCL recognized by the 2008 WHO classification [202], the T zone lymphoma (TZL) displays mainly CD4+ phenotype [318], while no consensus seems to exist regarding the phenotype of Lennert lymphoma, also known as lymphoepithelioid lymphoma (LHL), as according to some authors the most frequently expressed antigen is CD4 [327], while according to others it is CD8 [328,329]. In comparison to CD8+ cases, LHL CD4+ cases fare better from a prognostic point of view [325].

CD4 is seldom expressed on the cells of extranodal lymphomas [330], and as a rule is always missing on the cells of lymphomas with gamma/delta TCR, with only two exceptions reported in literature [331,332].

In accordance with the fact that neoplastic diseases of mature T cells very often display an abnormal T-related antigen expression, CD4 may be missing or display an abnormally low or high expression [333]; this behavior is frequently found in Sézary syndrome (SS) [94,97], but it has also been reported in angioimmunoblastic T cell lymphoma (AITL) [319] and in peripheral T cell lymphoma, not otherwise specified (PTCLnos) [94].

As for the neoplastic diseases of mature NK cells, CD4 has been reported in isolated cases of extranodal NK/T lymphoma (ENKL) nasal-type [334], and in a case of aggressive NK leukemia (ANKL) [335].

CD4 in myelodysplastic and chronic myeloproliferative diseases

CD4 has been demonstrated with flow cytometric techniques on the surface of the granulocytes of a patient affected by myelodysplasia characterized by the presence of translocation t(5;12); in this case it was hypothesized that the breakage of chromosome 12 was able to upregulate the expression of the antigen, encoded by a gene in 12p12 [336]. With flow cytometric techniques, CD4 has been reported on the mast cells of a case of mast cell leukemia [337].

CD4 in other pathological conditions

CD4 has been demonstrated with immunohistochemical techniques on the cells of Langerhans cell histiocytosis (LCH) [17], histiocytic sarcoma, also known as true histiocytic lymphoma (THL) [338,339], and in isolated cases of tumor of the indefinite cells (ICT), a solid neoplasm of the dendritic cells [40]. CD4 has been demonstrated with immunohistochemical techniques in an isolated case of follicular dendritic cell sarcoma (FDCS) [340].

CD5 Antigen

General features

CD5 is a 67 kD glycoprotein which is encoded by a gene situated on the long arm of chromosome 11 [341]. It is a T-related antigen that appears during T lymphocyte maturation, and it is normally expressed after the prothymocyte level [43,130]. CD5 is expressed on most mature T lymphocytes, but not on all of them, as either a subset of T lymphocytes with TCR gamma/delta [342] or a little subset of T lymphocytes with TCR alpha/beta [343] does not express the antigen.

Expanded populations of CD3+ CD5− T lymphocytes have been reported in the peripheral blood of subjects who have undergone allogeneic bone marrow transplantation [344].

CD5 can be considered a T-associated antigen, but not a T-specific molecule, inasmuch as it is expressed on the membrane of a subset of peripheral blood B lymphocytes, ranging between 17% and 25% of all the B lymphocytes [345–347]. Furthermore, CD5 is expressed on mature hematogones [348] and on B lymphocytes in fetal spleen [346], in the mantle of the germinal center [349], and in thymus [350]; it is expressed on most B lymphocytes in the cord blood or in the peripheral blood of the newborn [346], and on most peripheral B lymphocytes after autologous or allogeneic bone marrow transplantation [7].

CD5 is usually not expected to be expressed on NK lymphocytes, but some isolated reports exist documenting the antigen on a NK cell subset in normal subjects [351], in subjects affected by pulmonary tuberculosis [351], in subjects affected by multiple myeloma and plasmacytoma [352], and in two subjects with NK lymphocytosis induced by Epstein–Barr virus reactivation [353]. CD5 has also been reported on a dendritic cell subset [120].

Cytometric features

The staining of peripheral normal lymphocytes with an anti-CD5 monoclonal antibody generates a positive histogram with a gaussian-like peak, clearly separated from the negative component, but rather broad because of a certain variability in antigen expression.

The histogram produced by the staining of alpha/beta T lymphocytes generates a histogram with a channel peak representing the presence of 57±7 E03 ABC [56], but the CD5 intensity of expression is progressively decreasing in gamma/delta T lymphocytes [260,342] (Fig. 1.14), in the CD5+ CD19+ neoplastic B lymphocytes of B-CLL [354], and in the CD5+ CD19+ normal B lymphocytes, which express the antigen at a level lower than 2 E03 ABC [56].

Figure 1.14 Gamma/delta T lymphocytes (red) tend to express CD5 at a lower intensity than alpha/beta T lymphocytes.

Among peripheral T lymphocytes, CD5 expression tends to vary according to the subset; evidence exists that CD5 expression on CD4+ cells is 1.5 times more intense than on CD8 bright+ cells, and twice as intense as on CD8 dim+ cells [58].

CD5 is also expressed dimly on CD3+ CD6− T lymphocytes [355], on alpha/beta intraepithelial T lymphocytes in the gut [356], and on most thymocytes [130].

Diagnostic features

CD5 in neoplastic diseases of B cell precursors

As a rule, CD5 is not expressed in neoplastic diseases of B cell precursors [69], but it has been reported sporadically in isolated cases [357].

CD5 in neoplastic diseases of T cell precursors

CD5 is generally expressed in neoplastic diseases of T cell precursors [287], but it can be missing in the most immature forms, also known as early T, pro/pre T or T-stem cell leukemias [64]. This is in agreement with the EGIL classification of T lymphoblastic leukemias, according to which CD5 is typically present in the T II, T III and T IV forms, but missing in the most immature, T I [26].

CD5 in acute myeloid leukemias

In the acute myeloid leukemias CD5 has been reported in less than 10% of cases [29]; CD5 expression seems related to the AML-M5a [73] and AML-M0 subtypes [358]; in the AML-M0 subtype, CD5 seems to correlate with hypertriploid chromosome number [358].

The expression of CD5 has been reported in a case of acute basophilic leukemia arisen in a subject affected by myelodysplastic syndrome (MDS) [359].

CD5 in neoplastic diseases of mature B cells

The presence of CD5 divides the neoplastic diseases of mature B cells into two groups, the first made up of CD5+ diseases including B cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL), the second comprising CD5- diseases and including virtually all the remaining forms. Of course, this distinction only has didactic value and there are many important exceptions. Moreover, it is possible that new CD5+ clinical entities exist, not yet recognized as such by current classification, as perhaps in the case of CD5+ DLBCL with primary splenic onset.

From an operative point of view, a percentage of CD5+ B cells greater than 35% has been considered indicative for B cell lymphoma in the analysis of a lymph node biopsy without demonstrable light chain restriction [360].

Traditionally CD5+ diseases

As mentioned above, CD5 expression is a typical trait of B cell chronic lymphocytic leukemia and mantle cell lymphoma (MCL) [361–364]. In comparison with B-CLL, the neoplastic lymphocytes of MCL express CD5 at a higher intensity; this point has been confirmed by either flow cytometric [365] or immunohistochemical [366] studies. Nevertheless, it must not be forgotten that isolated cases of CD5- MCL have been reported [364].

CD5 is expressed either by typical or atypical B-CLL [367]; in B-CLL its increased expression is correlated with deletion of the long arm of chromosome 13 [368]. CD5 is also expressed on the elements of B-CLL in plasmacytoid transformation [369]; in these cases CD5 expression can constitute a useful element in the differential diagnosis with the lymphoplasmacytic lymphoma [370].

In some surveys B cell prolymphocytic leukemia (B-PLL) expresses CD5 in 50–70% of the cases [371–373], but in others it is consistently negative for the antigen [365]; this discrepancy can probably be explained either by the fact that in some surveys the cases derived from a pre-existing B-CLL are merged together with cases arising "de novo" or by the fact that leukemized MCL can sometimes present prolymphocytoid morphology, consequently being confused with B-PLL [374].

Traditionally CD5− diseases

Marginal zone lymphoma (MZL), hairy cell leukemia (HCL), lymphoplasmacytic lymphoma (LPL), follicular lymphoma (FL) and plasma cell neoplasms are traditionally considered CD5−.

Nevertheless, it must not be forgotten that CD5 has been reported in isolated cases of splenic lymphoma with villous lymphocytes (SLVL) [375], in 25% of cases of splenic marginal zone lymphoma (SMZL) [376], in isolated cases of either MALT or non-MALT extranodal marginal zone lymphoma (ENMZL) [377–381], in rare cases of either primary cutaneous [382] or nodal [383] [384] follicular lymphoma (FL) and in rare cases of HCL [303,385–388].

CD5 has been demonstrated on lymphocytes [389–392] and plasma cells [389] in rare cases of lymphoplasmacytic lymphoma (LPL), and on neoplastic plasma cells in a case of mantle cell lymphoma (MCL) in plasmacytic transformation [393].

When present in usually negative lymphomas, CD5 carries an unfavorable prognostic significance [89], especially in MALT type lymphomas, where it is correlated with leukemization and dissemination to bone marrow and other sites [377,379]. The demonstration of CD5 on HCL cells is of some practical importance, because the antigen expression is related to resistance to alpha-interferon [387], but sensitivity to cladribine (2-chloro-2′-deoxyadenosine, 2-CdA) [388]. It is noteworthy that in the case reported by Usha and collaborators, CD5 was expressed by hairy cells in bone marrow, but not by hairy cells in peripheral blood [388].

Sporadically CD5+ diseases

Apart from Richter syndrome, in which it is expected [394], CD5 has been reported either with cytometric or immunohistochemical techniques in 10% of cases of diffuse large B cell lymphoma (DLBCL) [395,396].

CD5+ DLBCL is usually characterized by a particular monomorphic morphology, by frequent intravascular or sinusoidal infiltration, and by frequent expression of bcl-2 [396].

In DLBCL, the CD5 expression correlates with an unfavorable prognosis [397], and it has been demonstrated that CD5+ cases respond worse to rituximab [398].

The CD5 expression is particularly frequent in intravascular large B-cell lymphoma (IVBCL) [399,400], where it seems devoid of prognostic significance [401]; CD5 expression has been reported in a case of T cell-rich large B cell lymphoma (TCRBCL) [402], and in some cases of Burkitt lymphoma (BL) in leukemic phase [403].

CD5 in neoplastic diseases of mature T and NK cells

CD5 is generally expressed on the cells of the neoplastic diseases of mature T cells, but it can also be missing or expressed in an aberrant way [333].

An irregular expression of CD5 has been sporadically reported in adult T cell leukemia/lymphoma (ATLL) [404], angioimmunoblastic T cell lymphoma (AITL) [95], and peripheral T cell lymphoma, not otherwise specified (PTCLnos) [35,94,95], and it is frequently found in T cell large granular lymphocytic leukemia (T-LGL) with