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Chapter 22 : Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias

Author: Elisabeth Paietta
Content Type: Reference
Publication Year: 2006

Category: Immunology

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Abstract:

The first classification of the acute leukemias in 1976 relied exclusively on the evaluation of cell size, granularity, nuclear shape, cytoplasmic appearance, cytochemical reactions, and dysplastic features of cells surrounding the “leukemic blast.” In selected cases, researchers have even succeeded in not only elucidating but also reversing the oncogenic mechanism, such as in acute promyelocytic leukemia (APL). APL has become the paradigm for the ultimate goal of leukemia diagnosis: to be able to tell an oncologist what targeted therapy a patient is a candidate for, based on the detection of a specific genotype. Some antibodies invariably stain all cells from a given lineage but vary markedly in their intensity of staining between normal and malignant cells, suggesting variable antigen densities (e.g., those of CD20 and CD22 on normal versus chronic lymphocytic leukemia [CLL] B lymphocytes); for other antigens, the fraction of cells binding the antibody will contain the diagnostic information (e.g., the percentage of CD34 or CD117 cells in any acute leukemia). Acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), or chronic myelogenous leukemia (CML) tissues should be cultured for 24 h without mitogens before processing. Samples from CLL require stimulation by B-cell mitogens (B-cell CLL) or T-cell mitogens (T-cell CLL) for cell division to occur. The principle of targeted therapy, as attractive and promising as it is, requires much more understanding of transforming molecular events and perturbed signaling pathways than the simple administration of indiscriminately cytotoxic chemotherapy.

Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22
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Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias
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Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22
/content/10.1128/9781555815905.ch22.ch22fig01
FIGURE 1
Typical antigen profile of -positive APL (for all L- and V-form and many S-form cases and for all M3 and some M3v morphologies). The scattergram in the left upper corner characterizes all cells present by size (forward scatter [FSC]) and granularity (side scatter [SSC]). A gate is set around all cells of interest. In the contour plot to the right, the leukemic promyelocytes are gated on the basis of CD117 expression (R2), whereby cells are stained with CD117 conjugated to phycoerythrin-cyanin 5 (PE CY5); this CD117-PE CY5 antibody is added to every antibody combination so that gating can be limited to leukemic cells. All subsequent contour plots show antigen expression only on gated leukemic promyelocytes. Such or similar gating strategy should always be applied in order to eliminate the inclusion of normal cells in the evaluation of antigen expression patterns. In all contour plots, data along the axes reflect cells stained with antibodies conjugated to fluorescein isothiocyanate (FITC). Along the axes are data for cells stained with antibodies conjugated to phycoerythrin (PE). Any blasts, which bind both FITC- and PE-antibodies in a given combination, appear in the right upper quadrant of the contour plot. For each contour plot, single or double antibodies tested (in addition to CD117) are indicated; CD clusters for all antibodies tested are written into the contour plots. Fluorescence intensity, a measure of antigen density, is usually expressed by the mean fluorescence channel of the cell population stained with the specific antibody of interest (along the or axis) divided by the mean fluorescence channel of the negative isotype control (mean fluorescence intensity or mean fluorescence intensity ratio).
10.1128/9781555815905/f0206-01_thmb.gif
10.1128/9781555815905/f0206-01.gif
Image of FIGURE 1
FIGURE 1

Typical antigen profile of -positive APL (for all L- and V-form and many S-form cases and for all M3 and some M3v morphologies). The scattergram in the left upper corner characterizes all cells present by size (forward scatter [FSC]) and granularity (side scatter [SSC]). A gate is set around all cells of interest. In the contour plot to the right, the leukemic promyelocytes are gated on the basis of CD117 expression (R2), whereby cells are stained with CD117 conjugated to phycoerythrin-cyanin 5 (PE CY5); this CD117-PE CY5 antibody is added to every antibody combination so that gating can be limited to leukemic cells. All subsequent contour plots show antigen expression only on gated leukemic promyelocytes. Such or similar gating strategy should always be applied in order to eliminate the inclusion of normal cells in the evaluation of antigen expression patterns. In all contour plots, data along the axes reflect cells stained with antibodies conjugated to fluorescein isothiocyanate (FITC). Along the axes are data for cells stained with antibodies conjugated to phycoerythrin (PE). Any blasts, which bind both FITC- and PE-antibodies in a given combination, appear in the right upper quadrant of the contour plot. For each contour plot, single or double antibodies tested (in addition to CD117) are indicated; CD clusters for all antibodies tested are written into the contour plots. Fluorescence intensity, a measure of antigen density, is usually expressed by the mean fluorescence channel of the cell population stained with the specific antibody of interest (along the or axis) divided by the mean fluorescence channel of the negative isotype control (mean fluorescence intensity or mean fluorescence intensity ratio).

Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22
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/content/10.1128/9781555815905.ch22.ch22fig02
FIGURE 2
Differential antigen expression profiles of differentiated AMLs dependent upon the presence of the / fusion gene. The first panel demonstrates antigen expression in a case of differentiated AML with a normal karyotype (AML-M2), negative for /. A small percentage of blast cells was gated by CD117 staining (R2) and analyzed. In comparison, the second panel demonstrates a typical surrogate marker profile for /-positive AML. While this case represents a differentiated AML with FAB M2 morphology, many cases with / present with a rather undifferentiated phenotype, occasionally CD33 negative. Note the high expression of CD34, which is typical for this AML subtype, the absence of CD11a, the partial expression of CD56, and the weak albeit definite presence of CD19. One more observation of importance is that /-positive AML does not involve expression of CD7 on the leukemic myeloblasts. SSC, side scatter; FSC, forward scatter; R1, gating of all white cells excluding debris; R2, gating on CD117 leukemic myeloblasts within R1. TC, third color; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein. Antibody combinations are written as CDs into the contour plots.
10.1128/9781555815905/f0209-01_thmb.gif
10.1128/9781555815905/f0209-01.gif
Image of FIGURE 2
FIGURE 2

Differential antigen expression profiles of differentiated AMLs dependent upon the presence of the / fusion gene. The first panel demonstrates antigen expression in a case of differentiated AML with a normal karyotype (AML-M2), negative for /. A small percentage of blast cells was gated by CD117 staining (R2) and analyzed. In comparison, the second panel demonstrates a typical surrogate marker profile for /-positive AML. While this case represents a differentiated AML with FAB M2 morphology, many cases with / present with a rather undifferentiated phenotype, occasionally CD33 negative. Note the high expression of CD34, which is typical for this AML subtype, the absence of CD11a, the partial expression of CD56, and the weak albeit definite presence of CD19. One more observation of importance is that /-positive AML does not involve expression of CD7 on the leukemic myeloblasts. SSC, side scatter; FSC, forward scatter; R1, gating of all white cells excluding debris; R2, gating on CD117 leukemic myeloblasts within R1. TC, third color; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein. Antibody combinations are written as CDs into the contour plots.

Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22
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Tables

Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias
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Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22
/content/10.1128/9781555815905.ch22.ch22tab01
TABLE 1
Established surrogate marker profiles for genetic lesions in acute leukemia
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TABLE 1

Established surrogate marker profiles for genetic lesions in acute leukemia

Citation: Paietta E. 2006. Phenotypic Correlates of Genetic Abnormalities in Acute and Chronic Leukemias, p 201-214. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch22

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