GCSF-R in Myelodysplastic and Myeloproliferative Disorders
GCSF-R in Myelodysplastic and Myeloproliferative Disorders
The GCSF-R mediates granulocyte proliferation and differentiation in both normal and malignant hematopoiesis. The gene is located on chromosome 1p32–35 (CSF3R gene) and belongs to class I of the cytokine (hematopoietic) receptor superfamily. Human GCSF-R is composed of 813 amino acids with a molecular mass of 130,000 to 150,000 Da. The molecule comprises an immunoglobulin-like domain, a cytokine receptor homology domain, 3 fibronectin type III–like domains, and transmembrane and cytoplasmic domains. The GCSF-R exerts biologic effects via ligand (granulocyte colony-stimulating factor [G-CSF])–induced homodimerization of the receptor. The G-CSF/GCSF-R complex mediates survival, proliferation, and maturation of neutrophils and regulates granulopoiesis under both steady-state and stress conditions. Cell signaling via GCSF-R is mediated primarily by the Jak/STAT pathway, but the Ras/Raf/MAP kinase and PKB/Akt pathways are also involved in signal transduction.
Mutations of the GCSF-R gene have been well documented in congenital neutropenia as well as the associated predisposition to myelodysplasia and acute myeloid leukemia. A single-nucleotide polymorphism in the GCSF-R gene has also been linked to predisposition to high-risk MDS.
In the present study, we characterized maturation-related surface density and distribution of CD114 in normal hematopoiesis. Our interest was in providing further detail in blast maturation that would be useful in the phenotypic diagnosis of myelodysplastic and myeloproliferative disorders. Abnormalities in CD114 expression in leukemia cells, leukemic cell lines, and MDS have been reported by several groups.
The pattern of CD114 expression in normal hematopoiesis was similar to that reported in earlier studies using ligand binding detection methods. We found that CD114 expression is initiated on early blasts and increases in density through the late blast stage, with peak density occurring at the promyelocyte stage. We noted a subsequent decrease in CD114 density on the maturing granulocytes. Shinjo et al used fluorescently labeled G-CSF in flow cytometric analysis and, similar to our findings, reported the CD114 density pattern on myeloid precursors cells as follows: CD33−CD34+ (early blasts) < CD33+CD34+ (late blasts) < CD33+CD34− cells (promyelocytes). In contrast to our findings, they reported that CD114 expression density was highest on mature granulocytes. We measured CD114 expression density on ficolled bone marrow specimens containing only a small proportion of mature granulocytes. It is possible that CD114 density is higher on pure populations of fully mature granulocytes than on promyelocytes, but this seems unlikely given the observed density decrease on the post-promyelocyte stages that we observed. Shinjo et al also used ligand binding as a detection method, and it is possible that a change in receptor affinity could account for this discrepancy. Begley et al evaluated receptor expression via binding of I-labled murine G-CSF. Similar to our findings, using direct receptor detection methods, they reported the highest CD114 density on human promyelocytes.
We observed similar mean CD114 density values for the defined myeloid maturation stages in the MDS groups in comparison with the control group. The MDS group stages, however, exhibited higher variance in expression density. Kimura and Sultana also reported more variation in CD114 density on the CD34+ blast populations in MDS. They observed that higher-grade MDS cases showed the highest CD114 density variation (either increased or decreased) in comparison with the control group. During case analysis, we observed individual MDS cases with significantly increased or decreased CD114 density in comparison with the control group. We partitioned the MDS cases based on CD114 density on the CD34+ blast population but observed no relationship to WHO MDS subtype (not shown).
In the CML cases, we observed significantly decreased mean CD114 density on early blasts in comparison with both the control and MDS groups but not on the later myeloid maturation stages. Lee et al performed flow cytometric analysis of peripheral blood samples in CML using an anti–CD114 antibody. They compared receptor density on mature granulocytes and monocytes in CML with a normal control group and reported a statistically significant lower mean CD114 receptor density on the CML granulocytes but not on the monocytes. They did not, however, evaluate earlier stages of myeloid differentiation. We observed lower mean CD114 density at all differentiation stages in the CML group in comparison with the control and MDS groups. In contrast to Lee et al, when comparing the control and CML groups, the smallest difference we observed was for CD114 density on the granulocytes.
In our control group, we observed that approximately 40% of the CD34+ blasts were expressing CD114. Yue et al reported a similar distribution in normal bone marrow, with 39% of blasts positive for CD114 expression. In contrast, Xu et al reported a significantly lower percentage, with only 21% of CD34+ blasts expressing CD114. In the MDS groups, we observed a similar mean percentage of CD34+ blasts expressing CD114 in comparison with the control group but with a higher variance. Yue et al reported a lower percentage of CD114+ blasts in the MDS cases in comparison with their control group, while Xu et al reported a higher percentage. The reported differences were not statistically significant in either study.
Neither Xu et al nor Yue et al evaluated the distribution of CD114+ blasts between the CD33− and CD33+ blast subsets. We found no significant difference in the mean distribution of CD114+ blast subsets comparing the CD33+ MDS subgroup with the control group. We did, however, note that due to the higher variance in the MDS group, the observable patterns of CD114+ blast subset distributions were informative in a significant number of MDS cases.
In the CML group, we found that the percentage of CD34+ blasts expressing CD114 was approximately 50% of the control group, but this did not quite reach statistical significance. This reduced percentage in the CML group was, however, significantly different from the combined MDS group and the CD33− MDS subgroup. In addition, the CML cases exhibited delayed CD114 receptor acquisition, as evidenced by statistically significant differences in the distribution of CD114-expressing blasts between the early and late blast compartments in comparison with both the control group and the CD33+ MDS subgroup.
In CML, the BCR-Abl fusion protein is associated with inhibition of GCSF-R (CD114) expression in response to G-CSF stimulation. Several groups have demonstrated that BCR-Abl inhibits expression of the C/CAAT/enhancer binding protein transcription factors C/EBPα and C/EBPε, which are responsible for upregulation of GCSF-R transcription. C/EBPα knockout mice display a complete differentiation arrest of the myeloid series, and C/EBPε knockout mice show aberrant granulocyte differentiation due to the inhibition of CD114 expression.
We observed a decreased percentage of CD114-positive blasts, delayed CD114 receptor acquisition, and decreased mean receptor density in the 5 CML cases evaluated. We observed a unique blast maturation abnormality involving CD33, CD34, and CD114, which was corroborated in an additional 6 historic cases of CML. Furthermore, the abnormal CD33 and CD34 maturation profile reverted to a normal pattern following suppression of the BCR-Abl–positive clone with Gleevec therapy. These observations indicate that the blast dysmaturation profile in CML is likely linked to the documented inhibition of GCSF-R expression by BCR-Abl. In practice, the CML blast pattern in bone marrow was 100% specific and 83% sensitive in the diagnosis of CML when cases of nonlymphoid leukocytosis were evaluated. Sensitivity in CML remission assessment was as good as morphology but clearly no substitute for quantitative rtPCR.
Drawbacks of this study include the small number of cases evaluated for GCSF-R expression and the absence of low-grade MDS cases such as refractory anemia (RA). There was a selection bias to collect higher-grade cases with more evident phenotypic abnormalities in the study design. Low-grade MDS cases are difficult diagnostically, and our experience is that only a small percentage of RA cases have sufficient phenotypic abnormalities to be unambiguously identified as low-grade MDS, even by highly trained analysts. We suspect that significant abnormalities of GCSF-R expression in RA would, like other phenotypic abnormalities, be of low frequency, but that remains to be demonstrated.
High-resolution analysis of CD34+ blast maturation can significantly enhance flow cytometric detection of myelodysplastic and myeloproliferative disorders. The GCSF-R (CD114) has a characteristic maturation-related expression profile in granulopoiesis and can provide additional phenotypic detail in flow cytometric assessment of CD34+ precursor maturation.
Discussion
The GCSF-R mediates granulocyte proliferation and differentiation in both normal and malignant hematopoiesis. The gene is located on chromosome 1p32–35 (CSF3R gene) and belongs to class I of the cytokine (hematopoietic) receptor superfamily. Human GCSF-R is composed of 813 amino acids with a molecular mass of 130,000 to 150,000 Da. The molecule comprises an immunoglobulin-like domain, a cytokine receptor homology domain, 3 fibronectin type III–like domains, and transmembrane and cytoplasmic domains. The GCSF-R exerts biologic effects via ligand (granulocyte colony-stimulating factor [G-CSF])–induced homodimerization of the receptor. The G-CSF/GCSF-R complex mediates survival, proliferation, and maturation of neutrophils and regulates granulopoiesis under both steady-state and stress conditions. Cell signaling via GCSF-R is mediated primarily by the Jak/STAT pathway, but the Ras/Raf/MAP kinase and PKB/Akt pathways are also involved in signal transduction.
Mutations of the GCSF-R gene have been well documented in congenital neutropenia as well as the associated predisposition to myelodysplasia and acute myeloid leukemia. A single-nucleotide polymorphism in the GCSF-R gene has also been linked to predisposition to high-risk MDS.
In the present study, we characterized maturation-related surface density and distribution of CD114 in normal hematopoiesis. Our interest was in providing further detail in blast maturation that would be useful in the phenotypic diagnosis of myelodysplastic and myeloproliferative disorders. Abnormalities in CD114 expression in leukemia cells, leukemic cell lines, and MDS have been reported by several groups.
The pattern of CD114 expression in normal hematopoiesis was similar to that reported in earlier studies using ligand binding detection methods. We found that CD114 expression is initiated on early blasts and increases in density through the late blast stage, with peak density occurring at the promyelocyte stage. We noted a subsequent decrease in CD114 density on the maturing granulocytes. Shinjo et al used fluorescently labeled G-CSF in flow cytometric analysis and, similar to our findings, reported the CD114 density pattern on myeloid precursors cells as follows: CD33−CD34+ (early blasts) < CD33+CD34+ (late blasts) < CD33+CD34− cells (promyelocytes). In contrast to our findings, they reported that CD114 expression density was highest on mature granulocytes. We measured CD114 expression density on ficolled bone marrow specimens containing only a small proportion of mature granulocytes. It is possible that CD114 density is higher on pure populations of fully mature granulocytes than on promyelocytes, but this seems unlikely given the observed density decrease on the post-promyelocyte stages that we observed. Shinjo et al also used ligand binding as a detection method, and it is possible that a change in receptor affinity could account for this discrepancy. Begley et al evaluated receptor expression via binding of I-labled murine G-CSF. Similar to our findings, using direct receptor detection methods, they reported the highest CD114 density on human promyelocytes.
We observed similar mean CD114 density values for the defined myeloid maturation stages in the MDS groups in comparison with the control group. The MDS group stages, however, exhibited higher variance in expression density. Kimura and Sultana also reported more variation in CD114 density on the CD34+ blast populations in MDS. They observed that higher-grade MDS cases showed the highest CD114 density variation (either increased or decreased) in comparison with the control group. During case analysis, we observed individual MDS cases with significantly increased or decreased CD114 density in comparison with the control group. We partitioned the MDS cases based on CD114 density on the CD34+ blast population but observed no relationship to WHO MDS subtype (not shown).
In the CML cases, we observed significantly decreased mean CD114 density on early blasts in comparison with both the control and MDS groups but not on the later myeloid maturation stages. Lee et al performed flow cytometric analysis of peripheral blood samples in CML using an anti–CD114 antibody. They compared receptor density on mature granulocytes and monocytes in CML with a normal control group and reported a statistically significant lower mean CD114 receptor density on the CML granulocytes but not on the monocytes. They did not, however, evaluate earlier stages of myeloid differentiation. We observed lower mean CD114 density at all differentiation stages in the CML group in comparison with the control and MDS groups. In contrast to Lee et al, when comparing the control and CML groups, the smallest difference we observed was for CD114 density on the granulocytes.
In our control group, we observed that approximately 40% of the CD34+ blasts were expressing CD114. Yue et al reported a similar distribution in normal bone marrow, with 39% of blasts positive for CD114 expression. In contrast, Xu et al reported a significantly lower percentage, with only 21% of CD34+ blasts expressing CD114. In the MDS groups, we observed a similar mean percentage of CD34+ blasts expressing CD114 in comparison with the control group but with a higher variance. Yue et al reported a lower percentage of CD114+ blasts in the MDS cases in comparison with their control group, while Xu et al reported a higher percentage. The reported differences were not statistically significant in either study.
Neither Xu et al nor Yue et al evaluated the distribution of CD114+ blasts between the CD33− and CD33+ blast subsets. We found no significant difference in the mean distribution of CD114+ blast subsets comparing the CD33+ MDS subgroup with the control group. We did, however, note that due to the higher variance in the MDS group, the observable patterns of CD114+ blast subset distributions were informative in a significant number of MDS cases.
In the CML group, we found that the percentage of CD34+ blasts expressing CD114 was approximately 50% of the control group, but this did not quite reach statistical significance. This reduced percentage in the CML group was, however, significantly different from the combined MDS group and the CD33− MDS subgroup. In addition, the CML cases exhibited delayed CD114 receptor acquisition, as evidenced by statistically significant differences in the distribution of CD114-expressing blasts between the early and late blast compartments in comparison with both the control group and the CD33+ MDS subgroup.
In CML, the BCR-Abl fusion protein is associated with inhibition of GCSF-R (CD114) expression in response to G-CSF stimulation. Several groups have demonstrated that BCR-Abl inhibits expression of the C/CAAT/enhancer binding protein transcription factors C/EBPα and C/EBPε, which are responsible for upregulation of GCSF-R transcription. C/EBPα knockout mice display a complete differentiation arrest of the myeloid series, and C/EBPε knockout mice show aberrant granulocyte differentiation due to the inhibition of CD114 expression.
We observed a decreased percentage of CD114-positive blasts, delayed CD114 receptor acquisition, and decreased mean receptor density in the 5 CML cases evaluated. We observed a unique blast maturation abnormality involving CD33, CD34, and CD114, which was corroborated in an additional 6 historic cases of CML. Furthermore, the abnormal CD33 and CD34 maturation profile reverted to a normal pattern following suppression of the BCR-Abl–positive clone with Gleevec therapy. These observations indicate that the blast dysmaturation profile in CML is likely linked to the documented inhibition of GCSF-R expression by BCR-Abl. In practice, the CML blast pattern in bone marrow was 100% specific and 83% sensitive in the diagnosis of CML when cases of nonlymphoid leukocytosis were evaluated. Sensitivity in CML remission assessment was as good as morphology but clearly no substitute for quantitative rtPCR.
Drawbacks of this study include the small number of cases evaluated for GCSF-R expression and the absence of low-grade MDS cases such as refractory anemia (RA). There was a selection bias to collect higher-grade cases with more evident phenotypic abnormalities in the study design. Low-grade MDS cases are difficult diagnostically, and our experience is that only a small percentage of RA cases have sufficient phenotypic abnormalities to be unambiguously identified as low-grade MDS, even by highly trained analysts. We suspect that significant abnormalities of GCSF-R expression in RA would, like other phenotypic abnormalities, be of low frequency, but that remains to be demonstrated.
High-resolution analysis of CD34+ blast maturation can significantly enhance flow cytometric detection of myelodysplastic and myeloproliferative disorders. The GCSF-R (CD114) has a characteristic maturation-related expression profile in granulopoiesis and can provide additional phenotypic detail in flow cytometric assessment of CD34+ precursor maturation.
