Lineage Uncommitted Antigens

Many lineage-uncommitted antigens are stem cell antigens, which in normal hematopoiesis mark cells with self-renewal capacity and differentiation potential along more than one cell lineage. During normal hematopoiesis, their expression diminishes parallel to progressive maturation. When present on leukemic blast cells, they may suggest a less differentiated phe-notype. Although their expression in leukemia does not support a particular lineage affiliation, some of these antigens demonstrate lineage preferences, for example, CD117. Because of their paucity in normal bone marrow and particularly peripheral blood, they are extremely useful in the monitoring of residual disease. Typical representatives are CD133 (the earliest of all known hematopoietic antigens and essential for bone marrow engraftment), CD34, HLA-DR, CD117 (c-kit), and CD90 (Thy-1), as well as the diagnostically less commonly used CD135 (FLT-3), CD115 (FMS), and CD105 (endoglin).64,113-121

CD133 is a transmembrane cell surface glycoprotein with homology to mouse prominin.122,123 Its distribution is limited to bone marrow. Normal circulating peripheral blood components116 and nonhematopoietic tissues fail to express the CD133 antigen, although CD133 transcripts were detected in some organs.123 A small portion of CD133+ bone marrow progenitor cells express CD135, HLA-DR, CD45, CD38lowbut lack CD34, CD117, and any evidence of lineage affiliation (lin-),124 which supports the concept that CD133+ cells give rise to

CD34+ cells121 and that only CD34+/CD133+ cells have repop-ulating capability, whereas CD34+/CD133- cells do not.116,119,125 Furthermore, CD133+ progenitor cells exhibited potential to differentiate into endothelial cells.126,127 Although CD133+, CD34+ bone marrow cells appear to be enriched predominantly among primitive, multilineage, and myeloid progenitor cells,124 they also contain a minor population of ery-throid burst-forming units116 as well as CD19+ or CD10+ B-lymphoid precursor cells.128 Because of its relatively recent discovery, the literature on CD133 expression in acute leukemia is somewhat contradictory, probably because most data are based on small numbers of patients. Snell et al.129 in 1998 suggested that CD133 could not be found in ALL. Since then, others have demonstrated CD133 in three of six patients with early Pre-B ALL, one patient tested with Pro-B-ALL, and one of two patients with T-ALL.128 In our experience in 155 patients with ALL, CD133 was expressed strongly and on more than 95 percent of blast cells in 71 percent of patients with Pro-B ALL, in 22 percent of patients with Early Pre-B ALL (with weak intensity in another patient), and in 7 percent of patients with Pre-B-or mature B-ALL (all with weak fluorescence intensity). Among 25 patients with T-ALL, only two demonstrated weak staining with CD133 on ALL blast cells (Paietta E, unpublished observation). Summarized data in AML suggest that CD133 expression is independent of FAB subtype, surface antigen expression pattern, cytogenetics, or age and without prognostic signifi-cance.128,130-132 In our experience, APL cells are consistently negative for CD133.18 Despite a close correlation between CD133 and CD34 in most studies, CD133 can be seen on blast cells lacking the CD34 antigen,133,134 particularly in undifferentiated, Pro-B ALL (8 of 15 CD133+ patients were CD34low/-) (Paietta E, unpublished observation).

CD34+ progenitors represent a heterogenous population of cells, the majority of which are already committed to the lym-phoid, myeloid, erythroid, or megakaryocytic cell lineage, as demonstrated by the expression of lineage-committed antigens (lin) together with the major histocompatibility complex (MHC) class II molecule HLA-DR and the CD38 antigen. Less than 1 percent of CD34+ cells are CD38low/-HLA-DR-lin-135,136 but express CD133, CD90, CD117, and CD135 as well as CD164, the last representing a new mucin-like molecule with negative regulatory function on CD34+ cell proliferation.137 These progenitors with the morphology of primitive blast cells differ from their descendants, the clonogenic cells, such as the multilineage progenitors CFU-GEMM (granulocyte-erythroid-macrophage-megakaryocyte CFU and CFU-Mix) and the lineage-committed BFU-E (erythroid burst-forming units), BFU-MK (megakaryocyte burst-forming unit), and CFU-GM (granulocyte-monocyte CFU), by their relative insensitivity to chemotherapy.136 The dull staining of stem cells with rho-damine123 reflects the presence of high levels of P-glycoprotein, the multidrug resistance (MDR) -mediating drug pump, causing increased cellular efflux of the dye.138 Analogous to this situation is the significant correlation between CD34 expression in AML and P-glycoprotein function.138,139

In children140 and adults,141,142 CD34+ B-lineage ALL is more likely to have an Early Pre-B (CD10+c^-) or Pro-B

(CD19+CD10-) ALL immunophenotype. In pediatric B-cel! ALL, CD34 correlates with good prognostic factors, such as hyperdiploidy and low incidence of central nervous system disease, whereas Early Pre-B ALL in adults is often associated with poor prognostic factors, such as older age and the Philadelphia chromosome (Ph).142>143 In Ph-negative adult ALL, CD34 has no prognostic impact.142 It should be mentioned, however, that CD34 determination is not part of many large clinical trials in adult ALL. CD34 is also common on the c^+s^+ blasts in transitional Pre-B-ALL, a subtype with better prognosis than Pre-B ALL in general.145 In pediatric T-cell ALL, CD34 correlates with central nervous system disease and other poor prognostic features.140

In pediatric as well as adult AML, the prognostic significance of CD34 ranges from irrelevant to predictive for failure to respond or decreased disease-free and overall survival. Such marked discrepancies reflect either differences in data interpretation, in particular, wide-ranging arbitrary cutoff points in the determination of CD34 positivity of a given leukemic cell population, or heterogeneity in patient populations. In fact, CD34 expression in AML is associated with a variety of poor-risk features, such as an immature phenotype,15,146,147 functional P-glycoprotein,139,148 and a higher incidence of chromosome 5 and 7 abnormalities.139,149,150 There is indication that only patients whose blast cells stain brightly for CD34 have a poor prognosis, whereas dim CD34 staining is prognostically equivalent to CD34 negativity.151 CD34 staining intensity varies dramatically depending on the fluorochrome used (PE-conjugated CD34 antibodies stain more strongly than FITC conjugates). Furthermore, there are three epitopes contained in the CD34 molecule (classes I, II, and III), whose distribution varies with the stage of cell maturation,152,153 making the choice of antibody crucial. Intracellular stores of CD34 release protein to the cell surface on extracellular stimulation.154 This provides an additional source of antigen staining for those laboratories choosing to fix the cells prior to antibody addition. All of these variables contribute to the fact that the prognostic significance of CD34, as a single antigen, is undetermined.

Aside from hematopoietic progenitor cells, the sialomucin CD34 is also expressed on endothelial cells, where it mediates binding of CD62L (L-selectin) on circulating leukocytes.136,155 A soluble form of CD34 is released from CD34+ cultured cells, and the same may occur during maturation in vivo.156

A small fraction of CD133+CD34+CD38low normal hematopoietic stem cells with long-term repopulating potential express CD90 (Thy-1),64,157,158 a member of the immunoglob-ulin superfamily, which is preferentially expressed on endothe-lial cells. Other human CD90+ hematopoietic tissues include a small percentage of fetal thymocytes and less than 1 percent of CD3+CD4+ circulating T lymphocytes.64 Expression of CD90 in AML is rare,159 and its frequency in ALL is poorly documented. Lamkin et al160 reported in childhood B-lineage ALL a CD34+Thy-1low phenotype in 65 percent of 49 children tested. To the contrary, we have observed among 230 adult patients with ALL only one case of Early Pre-B ALL with CD90+ CD34+CD133- blast cells, one case of undifferentiated Pro-B ALL with CD90lowCD34lowCD133+ blast cells, and one case of Pre-B ALL with CD90lowCD34-CD133- blast cells (Paietta E, unpublished observation). Aside from demonstrating a low incidence of CD90 in adult ALL (1.3 percent), our findings also suggest discordance in the expression pattern of stem cell antigens in acute leukemia.

Among normal mononuclear bone marrow cells, approximately two-thirds of CD34+ precursor cells express CD117 (c-kit), the stem cell factor or mast cell growth factor or steel factor receptor.161 Human CD117+CD34+lin- stem cells are capable of reconstituting hematopoiesis.162 Human precursors of all hematopoietic cell lineages exhibit CD117, with its density of expression being correlated with the stage of maturation. Among terminally differentiated hematopoietic cells, mast cells retain CD117 expression, as does a small fraction of natural killer cells expressing high levels of CD56.161,163 In contrast to this broad distribution of CD117 among normal precursor cells, among the acute leukemias it is preferentially expressed in AML.34-39 CD117 positivity in the absence of CD34 and HLA-DR expression is a typical feature of APL.35,164 In non-APL AML, CD117 is more common in phenotypes without a mono-cytic component35,38,39 and may be present in the absence of CD34.165 There are no proven associations between CD117 expression in non-APL AML and cytogenetic abnormalities or outcome in adult or pediatric AML.35-39,166 Detection of CD117 in ALL is controversial but appears to be rare and restricted to T-lineage ALL when the largest existing studies are consid-ered36,37,167-169 (Paietta E, unpublished observation).

CD109, another marker of hematopoietic stem cells, is also expressed on activated T lymphocytes, activated platelets, and endothelial cells.64,170 In combination with CD34, it is a marker of megakaryocytic precursor cells. Data in acute leukemia are sparse, with some indication that CD109 may be found in T-lineage ALL as well as in megakaryoblastic leukemia.64

CD105 (endoglin) expression on circulating CD133+ CD34+CD117+CD90+CD38low/- primitive progenitor cells marks a subset with potential of generating megakaryocytic precursors.171 As a component of the receptor for transforming growth factor P1 (TGF-P1), it may be involved in the autocrine control of megakaryocytopoiesis by TGF-P1. It is predominantly expressed by endothelial cells, and within the hematopoietic system it is found on early B cells in fetal bone marrow and a fraction of activated monocytes. A study on 318 patients with a variety of hematologic malignancies found CD105 expression in the majority of early B-lineage but not T-lineage ALL cases and in all types of AML cases except APL.172

HLA-DR is another marker used to aid in the characterization of hematopoietic stem cells. The CD34++HLA-DR+CD38low/-lin- fetal and adult bone marrow cells contain uncommitted hematopoietic progenitor cells and give rise to each of the hematopoietic cell lineages in the presence of hematopoietic growth factors.114 Along the myeloid lineage, its expression decreases with maturation, whereas it is present on the surface of both immature and mature monocytic cells. HLA-DR is expressed through all developmental stages of B lymphocytes and is only lost at the stage of the terminally differentiated plasma cell. During T-lymphopoiesis, HLA-DR is transiently expressed in the thymus and lost with differentiation. Occurrence of HLA-DR in AML and ALL follows the nor-

mal distribution. In T-ALL, HLA-DR expression by a subgroup of cases no longer appears of any clinical concern.

Differential expression of CD45 isoforms, derivatives of mRNA splice variants for the leukocyte common antigen complex, a family of transmembrane-type protein tyrosine phosphatases,64 have been used to segregate early from committed lymphoid and myeloid CD34+ progenitor cells.173-175 Precursors to CFU-Mix are included in the CD34+CD45RO+ CD45RA-CD90+CD38lowCD117lowrhodamine123du11 cell population,113 whereas CD34+CD45RA++ cells represent progenitors of the granulocyte-monocyte lineage, and CD34+ CD45RAlowCD71+ progenitors give rise to BFU-E.174,176 Evidence for the granulomonocyte committment of CD34+HLA-DR+++CD45RA++ cells is the intracytoplasmic expression of the myeloid-specific myeloperoxidase protein and the surface expression of the myeloid antigens CD33177 and CD64.178 Various mature leukocyte subpopulations express characteristic CD45 isoforms or combinations thereof.64 Using antibodies that bind to all CD45 isoforms, CD45 is ubiquitously found on blast cells from all AML subtypes. Caldwell et al.179 demonstrated that whereas normal myeloid stages predominantly express isoform CD45RO on the cell surface, most cases of AML express CD45RA, with or without CD45RO. In pediatric as well as adult ALL, 15 to 20 percent of B-lineage ALL cases lack CD45 expression. Absent CD45 expression in childhood ALL correlates with favorable outcome in association with chromosomal hyperdiploidy.180 The intensity level of CD45 staining appeared as an independent prognostic parameter in a large study of children with B-precursor ALL, with patients having bright CD45 faring the worst.181

Stages of progressive differentiation that have lost the ability to sustain prolonged hematopoiesis are associated with a decrease in stem-cell antigen expression concomitant with bright expression of CD38 and lineage-associated markers such as CD33 (myeloid lineage), CD71 (erythroid lineage), or early lymphoid antigens.114,182 Whereas CD38- cells have the characteristics of primitive blasts, CD38+ cells are blasts with differentiation features of multiple-cell lineages. Thus, the CD38 antigen is a marker for multilineage commitment of bone marrow progenitor cells. It is ubiquitously expressed on more differentiated cell types and is inducible by lymphocyte activa-tion.64 Expression of CD38 is ubiquitous in both ALL and AML. Low expression or lack of CD38 is a characteristic feature of the leukemic lymphocytes of B-cell chronic lymphocytic leukemia.2 Parallel with CD38, the CD7 T-cell molecule is increasingly expressed as another early marker of multipotent commit-ment.183 This concept is compatible with the finding of CD34+CD7+CD19+ cells, hypothetically derived from CD34+CD7+CD19- progenitors, which may represent uncommitted (dual T- and B-lineage) lymphoid precursors,184,185 analogous to the CD34+CD19+ and CD10+ bilineage progenitors that express the T-cell antigen CD2 during fetal hematopoiesis.186 Gore et al.187 and Dworzak et al.188 convincingly demonstrated the existence of CD34+lin- or cytoplasmic CD79a+ cells that expressed the DNA polymerase terminal deoxynucleotidyl transferase (TdT) in adult and pediatric bone marrow. Some CD34+TdT+CD79a+CD7+ cells coexpressed CD33 but not myeloperoxidase or intracytoplasmic CD3,188 which confirms previous data of CD7 expression on CD34+ myeloid precursor cells in normal bone marrow.189 Therefore, cross-lineage antigen expression is a well-recognized phenomenon not only in malignant but also in normal hematopoiesis.

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