Info

Children

Hypodiploidy <45 chromosomes 1%

Others 22%

B-Cell Lineage

Hypodiploidy >50 chromosomes 25%

T-Cell Lineage

HOX11 10q24 E2A-PBX1

Hypodiploidy <45 chromosomes 1%

Hypodiploidy >50 chromosomes 25%

Fig. 14-2. Estimated frequency of specific genotypes of ALL in children. (Modified from Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004;350:1535-48.)

Fig. 14-2. Estimated frequency of specific genotypes of ALL in children. (Modified from Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004;350:1535-48.)

Light microscopy, cytochemistry, immunophenotyping, and cytogenetics are necessary studies to characterize leukemic subtypes.

Morphology

Light Microscopy

Certain morphologic criteria have been established to differentiate lymphoblasts from myeloblasts (Table 14-2). Acute lymphoblastic leukemia can be further subclas-sified according to the French-American-British (FAB) classification as L1, L2, and L3 morphologic types (Table 14-3).

Uncommon ALL subtypes:

1. B-cell ALL with L1 morphology: ALL patients have been described with lym-phoblasts having L1 morphology but displaying B-cell ALL immunopheno-type. The blast cells are TdT+ in some cases. L3 lymphoblasts generally express immunoglobulin on their cell surface, whereas L1 do not.

2. Transitional pre-B ALL: This is characterized by:

a. Blasts cells that may express cytoplasmic and surface heavy chains but not Ig k or X light chains, indicating that these cells are in transition between pre-B and B stages of differentiation b. Blast cells that lack FAB L3 morphology or chromosomal translocations associated with B-cell ALL

c. Very good clinical outcome.

Cytochemistry

The cytochemical characteristics of the various types of leukemia are listed in Table 14-4.

Immunology*

The putative immunologic classification and cellular characteristics of B-lineage ALL and T-cell ALL are shown in Figure 14-1.

A panel of antibodies is used to establish the diagnosis of leukemia and to distinguish among the immunologic subclones. The panel should include at least one marker that is highly lineage specific, for example, CD19 for B lineage, CD7 for T lineage, and CD13 or CD33 for myeloid cells. In addition, the use of cytoplasmic CD79a for early pre-B-cell lineage, cytoplasmic CD3 for T lineage, and cytoplasmic myeloperoxidase for myeloid cells can be helpful in differentiating unclear immunophenotypes.

Immunophenotype Distribution of Acute Lymphoblastic Leukemia

Pre-B-cell accounts for 80% of ALL cases and is subdivided on the basis of cytoplas-mic immunoglobulin into transitional pre-B or common ALL antigen (CALLA positive).

Mature B-cell type accounts for 1-2% of ALL cases. These have surface immunoglobulin positivity and are treated as Burkitt lymphoma. The prognosis has improved and is now similar to other subtypes of high-risk ALL. T-cell type accounts for 15-20% of ALL cases. This subtype is associated with:

• Older age at presentation

• High initial WBC count

*For CD antigen nomenclature, refer to Appendix 2.

• Presence of extramedullary disease

• Poor prognosis (treatment on high-risk intensive therapies has improved the prognosis).

Cytogenetics and Molecular Characteristics

The cytogenetic abnormalities observed in leukemia have biologic and prognostic significance. The realization of the biologic significance of leukemia cytogenetics has resulted in the application of molecular methods to understand various mechanisms of leukemogenesis. Specific cytogenetic abnormalities have been shown to have prognostic significance (Table 14-6).

Molecular Genetics of Leukemia of ALL

Table 14-5 lists and Figure 14-2 shows the distribution of molecular rearrangements and chromosomal translocations in childhood acute lymphoblastic leukemia. Seventy-five percent of childhood ALL cases have evidence of chromosomal translocations. The following translocations result in activation of protein kinases by onco-genes and activation of transcription factors:

1. Tel-AML1 fusion gene t(12;21)(p13q;q22). t(12;21) is detected by standard cyto-genetics in less than 1 in 1000 cases, whereas, using molecular techniques, it is detected in 25% of pre-B cases. This translocation is associated with an excellent prognosis.

2. BCR-ABL fusion gene t(9;22)(q34;q11). t(9;22) occurs in only 3-5% of pediatric ALL cases. This is in contrast to adult ALL where this translocation is present in 25% of cases and 95% of adult chronic myelogenous leukemia (CML) cases. In pediatric BCR-ABL-positive ALL, the BCR breakpoint produces a 190-kb protein (p190) in contrast to CML where a different protein (p210) is usually produced. The t(9;22) in pediatric ALL is usually associated with older age, higher WBC count, and frequent CNS involvement at diagnosis.

3. E2A-PBX1 fusion gene t(1;19)(q23;p13.3). t(1;19) is frequently associated with an elevated WBC at diagnosis and occurs in 25% of cases with a pre-B cytoplasmic immunoglobulin-positive phenotype. Intensive therapy is necessary in this subtype.

4. MLL gene rearrangement at chromosome band 11q23 affects 80% of ALL cases in infants, 3% of ALL cases in older children, and 85% of secondary AML

Table 14-6. Prognostic Significance of Chromosomal Abnormalities in ALL

Chromosomal abnormalities

Hyperdiploidy >50 chromosomes 47-50 chromosomes Near triploid, 66-73 chromosomes Near tetraploid, 82-94 chromosomes (clinical peculiarities: more often T-ALL; L2 morphology; expression of one or more myeloid antigens) Normal diploid, 46 chromosomes Hypodiploidy, <46 chromosomes Pseudodiploid t(1;19) t(4;11) t(9;22)

5-year event-free survival (confidence level)

Not known, but probably good

Not known, but probably less than 60%

involving the topoisomerase II inhibitor. This translocation carries a very poor prognosis with a survival of less than 20%, despite intensive therapy.

5. B-cell ALL translocations involve MYC genes on chromosome 8q24. Eighty percent of B-ALL cases contain t(8;14)(q24;q32); the remaining cases have t(2;8) (p12;q24) or t(8;22)(q24;q11). All of these translocations deregulate MYC expression and need to be treated intensively with the same agents used to treat Burkitt lymphoma.

Some examples of the prognostic significance of chromosomal abnormalities in ALL are listed in Table 14-6.

Prognostic Factors

Patients who are between ages 1 and 9 with an initial WBC <50,000/mm3 (standard risk), which includes two thirds of pre-B ALL patients, have a 4-year event-free survival of 80%. The remaining patients (high risk) have a 4-year event-free survival of 65%. Factors that should be included in risk classification are:

1. Age: Patients under 1 year of age and greater than 10 years of age have a worse prognosis than children >1 years and <10 years of age. Infants under 1 year of age have the worst prognosis.

2. White cell count: Children with the highest WBC tend to have a poor prognosis (Table 14-7).

3. Immunophenotype: Early pre-B-cell ALL has the best prognosis. Mature T-cell ALL has a worse survival due to its association with older age and higher WBC at diagnosis. Mature B-cell ALL previously had a poor prognosis with early relapses and CNS involvement but recent aggressive therapies have improved prognosis.

4. DNA index >1.16 hyperdiploid ALL with greater than 50 chromosomes has been associated with good outcome due to increased apoptosis and increased sensitivity to chemotherapeutic agents.

5. Cytogenetics: The combinations of trisomies of chromosomes 4, 10, and 17 have been associated with a very low risk of treatment failure and good outcome. Translocations involving the MLL rearrangement on 11q23 have been associated with a worse prognosis. The Philadelphia chromosome t(9;22)(q34;q11) ALL is the most difficult translocation to treat and has a bad prognosis. Hypodiploid ALL is also associated with a poor prognosis.

6. CNS disease: The presence of CNS disease at diagnosis is an adverse prognostic factor despite intensification of therapy with CNS irradiation and additional intrathecal therapy. The presence of blast on cytospin without an increased WBC (CNS2 status) is also associated with poor outcome.

7. Early response to induction therapy: Patients who are not in remission at the end of induction therapy have a very poor prognosis. Bone marrow results on day 7 and day 14 of induction therapy have also been used to estimate response to therapy. In future clinical trials the presence of minimal residual disease at day 28 of induction therapy will be used in addition to day 7 and day 14 bone marrow blasts percentages in determining rapid early response and subsequent therapy. Table 14-8 lists the classification of bone marrow remission status in ALL.

Table 14-7 lists a proposed risk classification system.

Future Directions in ALL Classifications

1. Gene expression profiling using DNA microassay technology can define biologically and prognostically distinctive ALL subsets, can identify genes that

Table 14-7. Proposed Risk Classification System of Pre-B-Cell ALL

Risk Group

Features

Low (treated same as standard risk)

Standard

High risk

Very high risk

Age >1 year to <10 years of age WBC count <50,000/mm3 tel-AML or trisomy 4, 10, 17 Age >1 year to <10 years of age WBC count <50,000/mm3 Not tel-AML or trisomy 4, 10, 17 Age >10 years WBC >50,000/mm3 CNS 3 or testicular disease MLL translocation Ph+ leukemia

Hypodiploidy < 45 chromosomes Induction failure

Table 14-8. Classification of Bone Marrow Remission Status in ALL

Classification

% Blasts in bone marrow

M1 bone marrow M2 bone marrow M3 bone marrow

<5% <25% >25%

Based on 200 cell count, true remission requires Mj marrow status with normal marrow cellularity and presence of all cell lines.

Based on 200 cell count, true remission requires Mj marrow status with normal marrow cellularity and presence of all cell lines.

may be responsible for leukemogenesis, and may identify genes for which targeted therapy could be developed.

2. Host pharmacogenomics: Gene polymorphisms for genes that encode drug-metabolizing enzymes can influence the efficacy and toxicity of chemotherapy.

a. Gene polymorphism for thiopurine methyltransferase (TPMT) is a gene that catalyzes the inactivation of mercaptopurine. Ten percent of the population carries at least one variant allele. This results in high levels of active metabolites of mercaptopurine. These patients, especially homozygous patients, have an increased risk of side effects and require marked reduction of 6-mer-captopurine doses.

b. Gene polymorphism for thymidylate synthetase (target of methotrexate) has been associated with increased enzyme expression and poor outcome in ALL.

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