Myeloid antigens in childhood lymphoblastic leukemia: clinical data point to regulation of CD66c distinct from other myeloid antigens

Abstract

Background: Aberrant expression of myeloid antigens (MyAgs) on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon, although its regulating mechanisms are unclear. MyAgs in ALL are interpreted e.g. as hallmarks of early differentiation stage and/or lineage indecisiveness. Granulocytic marker CD66c -- Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is aberrantly expressed on ALL with strong correlation to genotype (negative in TEL/AML1 and MLL/AF4, positive in BCR/ABL and hyperdiploid cases).

Methods: In a cohort of 365 consecutively diagnosed Czech B-precursor ALL patients, we analyze distribution of MyAg+ cases and mutual relationship among CD13, CD15, CD33, CD65 and CD66c. The most frequent MyAg (CD66c) is studied further regarding its stability from diagnosis to relapse, prognostic significance and regulation of surface expression. For the latter, flow cytometry, Western blot and quantitative RT-PCR on sorted cells is used.

Results: We show CD66c is expressed in 43% patients, which is more frequent than other MyAgs studied. In addition, CD66c expression negatively correlates with CD13 (p < 0.0001), CD33 (p = 0.002) and/or CD65 (p = 0.029). Our data show that different myeloid antigens often differ in biological importance, which may be obscured by combining them into "MyAg positive ALL". We show that unlike other MyAgs, CD66c expression is not shifted from the onset of ALL to relapse (n = 39, time to relapse 0.3-5.3 years). Although opposite has previously been suggested, we show that CEACAM6 transcription is invariably followed by surface expression (by quantitative RT-PCR on sorted cells) and that malignant cells containing CD66c in cytoplasm without surface expression are not found by flow cytometry nor by Western blot in vivo. We report no prognostic significance of CD66c, globally or separately in genotype subsets of B-precursor ALL, nor an association with known risk factors (n = 254).

Conclusion: In contrast to general notion we show that different MyAgs in lymphoblastic leukemia represent different biological circumstances. We chose the most frequent and tightly genotype-associated MyAg CD66c to show its stabile expression in patients from diagnosis to relapse, which differs from what is known on the other MyAgs. Surface expression of CD66c is regulated at the gene transcription level, in contrast to previous reports.

Figure 1

Correlation of ALL genotype categories and percentage of CD66c positivity. Median percentage of CD66c^pos blasts is listed below each genotype group. Data of consecutive unselected patients with BCP ALL (n = 373) are shown.

Figure 2

Graphical illustration of myeloid antigen positivity in childhood B-precursor ALL. For each antigen, positive cases are represented by a colored form. The areas of the forms roughly correspond to the frequency of positive cases (observed numbers of patients are marked in red) while the shapes are constructed to illustrate the respective coexpressions. An arbitrary cutoff value of 20% is used for all antigens. The CD66c positivity correlates with negativity of any of the following: CD33 (p = 0.002), CD13 (p < 0.0001) and CD65 (p = 0.029). There was a significant correlation between CD33 and CD13 positivity (p < 0.0001) and between CD15 and CD65 positivity (p = 0.0002) whereas the positivity of no other two antigens of the ones shown correlated significantly with each other. Total number of B-precursor cases illustrated is 365.

Figure 3

Relationship of surface and cytoplasmic expression of CD66c. Percentage of surface expression of CD66c in ALL blasts is plotted against cytoplasmic expression (after cell membrane permeabilization). Samples of 20 patients at ALL diagnosis are shown, 12 CD66c negative and 8 CD66c positive. Regression coefficient R² = 0.927

Figure 4

Transcription of CEACAM6 versus surface CD66c expression on sorted cells. FACSsorted CD66c surface negative (CD66c^neg) or positive (CD66c^pos) ALL lymphoblasts, five patients with heterogeneous CD66c expression were sorted into both CD66c negative and CD66c positive fraction (lines connect sorted fractions from the same specimen). Mann-Whitney test was used to compare groups (n = 32). CEACAM6n value is normalized to beta-2-microglobulin (see Methods).

Figure 5

Western blot of granulocytes, ALL samples of CD66c positive cases and surface CD66c^neg cell lines with TEL/AML1pos (REH), MLL/AF4pos (RS4;11) translocation and with no fusion (NALM-6).

Figure 6

Stability of CD66c from diagnosis to relapse. Each circle represents one patient (n = 39). Percentage of CD66c^pos blasts at diagnosis is plotted against percentage of CD66c^pos blasts at relapse. Regression line with 95% confidence R² = 0.755

Figure 7

Relapse free survival of cases with CD66c ^pos (blue line) or CD66c^neg(red line) B-precursor ALL. Unselected consecutive patients treated on ALL BFM95 protocol (median follow up 3.64 years). Since surface CD66c associates with genotype, separate analyses for distinct genotype subgroups are shown.