Contexto actual

Leukemic blast cells are identified at diagnosis in the cerebrospinalfluid (CSF) of approximately one third of children and 5–10% of adults with ALL, most of whom have no neurological symptoms. Although cen-tral nervous system (CNS) leukemia is defined by the presence of at leastfive leukocytes per μL of CSF and the detection of leukemic blast cells (Figure 7.4), or cranial nerve palsy (Table 7.3), the presence of any leukemic cells in the CSF (even from introduction due to a traumatic lumbar puncture) predicts an increased risk of relapse in children with ALL. In cases with borderline CSF leukocyte counts and/or uncertain morphological findings, additional tests may be necessary. Molecular genetic studies to detect specific gene fusions or antigen receptor gene re-arrangements could be helpful but are not routinely b

a c

Figure 7.2.Morphological features of ALL cells. Shown are examples according to the FAB classification. Bone marrow smears were stained with Wright–Giemsa (images courtesy of Dr. Mihaela Onciu, St. Jude Children’s Research Hospital, Memphis, TN).

b a

d c

f e

Figure 7.3.Bone marrow trephine of B-lineage ALL.

a. Hematoxylin and eosin (H&E) stained section showing the diffuse infiltrate of monomorphic blast cells.

b. Higher power view showing the cytological details and some pleomorphism of nuclear shape and the indistinct nucleoli.

c. The blast cells are CD34-positive, (d) CD10-positive, (e) CD79a-positive and (f) TdT-positive (immunoperoxidase and DAB substrate).

Images courtesy of Dr. Wendy Erber, Addenbrooke’s Hospital, Cambridge, UK.

performed. Flow cytometric analysis can also be infor-mative but requires a substantial number of cells.

Staining cytocentrifuge preparations with antibodies to terminal deoxynucleotidyl transferase (TdT) and observation by fluorescence microscopy is probably the most widely applicable method to clarify suspect CSF findings, although the method is tedious and time-consuming (Figure 7.4).

Immunophenotyping

B-lineage ALL

B-lineage ALL cells at any stage of maturation express CD19 antigen and, in almost all cases, have cytoplas-mic CD22 and CD79α. CD22 is prominent in the cytoplasm among the most immature cases [11].

Antigen expression can also be used to subtype cases, based on phenotypic differentiation (seeFigure 2.1), into early pre-B ALL, pre-B ALL and B-cell ALL.

Early pre-B ALL cases also express CD10 and TdT (approximately 90% of cases), and CD34 (more than 75% of cases). Early pre-B ALL cells lack expression of surface and cytoplasmic immunoglobulins [11]. CD20 is present in one-half of cases and its intensity can increase during treatment [14]. ALL cases with rearrangement of theMLL gene typically have an early pre-B ALL pheno-type with distinctive phenotypic features such as expres-sion of CD15, CD65 and chondroitin proteoglycan sulfate (NG2), and absence of CD10. Hyperdiploidy (chromosome number > 50) is typically associated with weak or undetectable CD45 expression [11].

Pre-B ALL is defined by the presence of cytoplas-mic immunoglobulinμ heavy chains in the lympho-blasts with no detectable surface immunoglobulins; in rare cases leukemic cells express both cytoplasmic and surface immunoglobulinμ heavy chains without κ or λ light chains [11]. Pre-B ALL cells usually express CD10 and TdT, with approximately two-thirds of cases also expressing CD34; CD20 expression is variable. The t(1;19)(q23;p13) or the der(19)t(1;19)(q23;p13) genetic abnormalities are found in 20–25% of pre-B ALL cases.

B-cell ALL is characterized by the expression of sur-face immunoglobulinμ heavy chains plus either κ or λ light chains [11]. Commonly, cells have L3 morphology

b

a c

Figure 7.4.CNS leukemia.

a. Cytocentrifuge preparation of cerebrospinalfluid (CSF) from a patient with ALL stained with Wright–Giemsa showing ALL blasts (image courtesy of Dr. Mihaela Onciu, St. Jude Children’s Research Hospital, Memphis, TN).

b. Cytocentrifuge preparation of CSF from another patient with ALL stained with anti-TdT (red; tetramethylrhodamine) and anti-CD3 (green;

fluorescein isothiocyanate) antibodies. TdT-positive ALL blasts (arrows) are admixed with normal T-cells.

c. The same microscopicfield as in b viewed by phase contrast microscopy; arrows point to the TdT-positive blasts.

Table 7.3.CNS status classification.

Status Leukocytes (per µL) Lymphoblasts

CNS1 <5 No

CNS2 <5 Yes

CNS3* ≥5 Yes

* Presence of cranial nerve palsies indicates CNS3 even with < 5 leukocytes per L and/or absent lymphoblasts.

(according to the FAB classification), express CD20, and frequently CD10; CD34 is negative. An uncommon sub-type of B-cell ALL is characterized by blast cells with L1 or L2 morphology, and expression of TdT and/or CD34.

T-lineage ALL

T-lineage ALL cells express CD7 and CD3 antigens, the latter most frequently only in the cytoplasm [11].

Other markers commonly expressed include CD2, CD5 and TdT; CD1a, surface CD3, CD4 and CD8 are detected in approximately 40% of cases. HLA-DR expression is uncommon, and 40–45% of cases are CD10⁺and/or CD21⁺. CD79α is weakly expressed in approximately one third of cases.

T-lineage ALL can be divided into three stages of immunophenotypic differentiation reflecting normal stages of thymic differentiation (seeFigure 2.2): early (CD7⁺, cCD3⁺, surface CD3⁻, CD4⁻ and CD8⁻), mid or common (cCD3⁺, surface CD3⁻, CD4⁺, CD8⁺ and CD1a⁺), and late (surface CD3⁺, CD1a⁻ and either CD4⁺ or CD8⁺) [11]. However, many cases

have immunophenotypic patterns that do not fit these thymic maturation stages. T-cell receptor (TCR) proteins are heterogeneously expressed in T-lineage ALL. In approximately two-thirds of cases, membrane CD3 and TCR proteins are absent. In half of these cases, however, TCR proteins (TCRβ, TCRα or both) are present within the cytoplasm.

Most cases with membrane CD3 and TCR chains express the TCRαβ, whereas a minority express TCRγδ proteins.

A subtype of T-ALL, named ETP-ALL, with the gene expression profile of normal early T-cell pre-cursor cells, has recently been identified [7]. This leukemia is derived from a population of recent immigrants from the bone marrow to the thymus and which retain multi-lineage differentiation poten-tial [7]. These leukemias are CD7- and CD3-positive (mostly only in the cytoplasm), lack CD1a and CD8 expression, have weak or absent CD5 expression and express at least one stem cell or myeloid-associated antigen (e.g. CD34, CD117, CD13, CD33, CD11b) (Figure 7.5). These cases are characterized by a

Figure 7.5.Immunophenotypic differences between T-ALL and ETP-ALL. Diagnostic bone marrow samples from a patient with T-ALL (top row) and two patients with ETP-ALL (middle and bottom rows) were analyzed byflow cytometry. Biexponential dot plots are shown.

dismal response to therapy and a high rate of relapse [7].