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The use of MAGE C1 and flow cytometry to determine the malignant cell type in multiple myeloma.

Wienand K, Shires K - PLoS ONE (2015)

Bottom Line: Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls.Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1.MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/-/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples.

View Article: PubMed Central - PubMed

Affiliation: Division of Haematology, University of Cape Town, Cape Town, South Africa.

ABSTRACT
The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/-/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34+/MAGE C1+ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells.

No MeSH data available.


Related in: MedlinePlus

Examples of flow cytometric analysis of the BM of a donor (A) and two MM patients (B and C) to determine the different B cell lineage cell populations expressing MAGE C1.Mononuclear cells from donor and MM patient BM were stained with different antibodies to determine the cell phenotype and stage of B cell maturation where MAGE C1 was expressed. A and B: Density plots (SS and CD45) were used to selectively gate around specific cell populations including plasma cells (R1), stem/immature B lymphocytes (R2) and mature B lymphocytes (R3). Using various antibodies specific to the cells of interest gated in R1, R2 and R3, cells with positive antibody expression were further selectively gated (R4) and MAGE C1 expression was determined with histograms. A: Donor BM indicated that all selected cell populations were negative for MAGE C1 expression. B: MAGE C1 expression was observed in the stem/immature B lymphocytes and not in the non-proliferating plasma or mature B lymphocytes of the MM patient. C: Density plots (SS and CD45) were used to selectively gate the plasma cells (R1) and these cells were further confirmed with CD138 (R2). The proliferation index of the plasma cells was determined with Ki-67 expression and this was correlated to MAGE C1 expression via histograms, respectively. MAGE C1 expression was only observed in the proliferating plasma cells (Ki-67>20%). 1: Plasma cells, 2: Stem/immature B lymphocytes, 3: Lymphocytes, 4: Residual monocytes, 5: Residual Granulocytes.
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pone.0120734.g001: Examples of flow cytometric analysis of the BM of a donor (A) and two MM patients (B and C) to determine the different B cell lineage cell populations expressing MAGE C1.Mononuclear cells from donor and MM patient BM were stained with different antibodies to determine the cell phenotype and stage of B cell maturation where MAGE C1 was expressed. A and B: Density plots (SS and CD45) were used to selectively gate around specific cell populations including plasma cells (R1), stem/immature B lymphocytes (R2) and mature B lymphocytes (R3). Using various antibodies specific to the cells of interest gated in R1, R2 and R3, cells with positive antibody expression were further selectively gated (R4) and MAGE C1 expression was determined with histograms. A: Donor BM indicated that all selected cell populations were negative for MAGE C1 expression. B: MAGE C1 expression was observed in the stem/immature B lymphocytes and not in the non-proliferating plasma or mature B lymphocytes of the MM patient. C: Density plots (SS and CD45) were used to selectively gate the plasma cells (R1) and these cells were further confirmed with CD138 (R2). The proliferation index of the plasma cells was determined with Ki-67 expression and this was correlated to MAGE C1 expression via histograms, respectively. MAGE C1 expression was only observed in the proliferating plasma cells (Ki-67>20%). 1: Plasma cells, 2: Stem/immature B lymphocytes, 3: Lymphocytes, 4: Residual monocytes, 5: Residual Granulocytes.

Mentions: To determine which cellular populations were expressing MAGE C1 in both the BM and PB, a series of antibody panels was used. The initial control donor investigations showed a high degree of non-specific binding of the secondary antibody for MAGE C1 to the monocytic and residual granulocytic populations, despite various blocking strategies. Using antibodies including CD14, CD13, and CD33 the monocytic and granulocytic populations were carefully determined and specific gates were established to exclude all cells contributing to false positivity [23]. Thus using specific gates as well as a combination of SS and CD45 expression to initially identify the cell subgroups of interest, our analysis focused on the plasma cell, stem cell/immature B lymphocyte populations, as well as the mature B lymphocytes (pre-germinal centre) [24]. Based on CD45 expression, bright MAGE C1 positive expression was associated with the stem/immature B lymphocyte cell region in both the MM BM and PB samples of all patients, while a second positive MAGE C1 subpopulation of proliferating plasma cells (dual Ki-67+/CD138+ phenotype) was observed in six patients in both the BM and corresponding PB sample, as well as three additional patients with unmatched PB/BM samples. This population was identified using a combination of antibody panels 2 and 4 to first selectively gate the proliferating plasma cells in the CD138+ population (distinct subpopulation) and then investigate their MAGE C1 expression. Fig. 1 shows examples of the two distinct populations observed in the BM. No significant MAGE C1 expression was observed in any of the donor or PB samples using this protocol, confirming a link to the MM diseased state.


The use of MAGE C1 and flow cytometry to determine the malignant cell type in multiple myeloma.

Wienand K, Shires K - PLoS ONE (2015)

Examples of flow cytometric analysis of the BM of a donor (A) and two MM patients (B and C) to determine the different B cell lineage cell populations expressing MAGE C1.Mononuclear cells from donor and MM patient BM were stained with different antibodies to determine the cell phenotype and stage of B cell maturation where MAGE C1 was expressed. A and B: Density plots (SS and CD45) were used to selectively gate around specific cell populations including plasma cells (R1), stem/immature B lymphocytes (R2) and mature B lymphocytes (R3). Using various antibodies specific to the cells of interest gated in R1, R2 and R3, cells with positive antibody expression were further selectively gated (R4) and MAGE C1 expression was determined with histograms. A: Donor BM indicated that all selected cell populations were negative for MAGE C1 expression. B: MAGE C1 expression was observed in the stem/immature B lymphocytes and not in the non-proliferating plasma or mature B lymphocytes of the MM patient. C: Density plots (SS and CD45) were used to selectively gate the plasma cells (R1) and these cells were further confirmed with CD138 (R2). The proliferation index of the plasma cells was determined with Ki-67 expression and this was correlated to MAGE C1 expression via histograms, respectively. MAGE C1 expression was only observed in the proliferating plasma cells (Ki-67>20%). 1: Plasma cells, 2: Stem/immature B lymphocytes, 3: Lymphocytes, 4: Residual monocytes, 5: Residual Granulocytes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368436&req=5

pone.0120734.g001: Examples of flow cytometric analysis of the BM of a donor (A) and two MM patients (B and C) to determine the different B cell lineage cell populations expressing MAGE C1.Mononuclear cells from donor and MM patient BM were stained with different antibodies to determine the cell phenotype and stage of B cell maturation where MAGE C1 was expressed. A and B: Density plots (SS and CD45) were used to selectively gate around specific cell populations including plasma cells (R1), stem/immature B lymphocytes (R2) and mature B lymphocytes (R3). Using various antibodies specific to the cells of interest gated in R1, R2 and R3, cells with positive antibody expression were further selectively gated (R4) and MAGE C1 expression was determined with histograms. A: Donor BM indicated that all selected cell populations were negative for MAGE C1 expression. B: MAGE C1 expression was observed in the stem/immature B lymphocytes and not in the non-proliferating plasma or mature B lymphocytes of the MM patient. C: Density plots (SS and CD45) were used to selectively gate the plasma cells (R1) and these cells were further confirmed with CD138 (R2). The proliferation index of the plasma cells was determined with Ki-67 expression and this was correlated to MAGE C1 expression via histograms, respectively. MAGE C1 expression was only observed in the proliferating plasma cells (Ki-67>20%). 1: Plasma cells, 2: Stem/immature B lymphocytes, 3: Lymphocytes, 4: Residual monocytes, 5: Residual Granulocytes.
Mentions: To determine which cellular populations were expressing MAGE C1 in both the BM and PB, a series of antibody panels was used. The initial control donor investigations showed a high degree of non-specific binding of the secondary antibody for MAGE C1 to the monocytic and residual granulocytic populations, despite various blocking strategies. Using antibodies including CD14, CD13, and CD33 the monocytic and granulocytic populations were carefully determined and specific gates were established to exclude all cells contributing to false positivity [23]. Thus using specific gates as well as a combination of SS and CD45 expression to initially identify the cell subgroups of interest, our analysis focused on the plasma cell, stem cell/immature B lymphocyte populations, as well as the mature B lymphocytes (pre-germinal centre) [24]. Based on CD45 expression, bright MAGE C1 positive expression was associated with the stem/immature B lymphocyte cell region in both the MM BM and PB samples of all patients, while a second positive MAGE C1 subpopulation of proliferating plasma cells (dual Ki-67+/CD138+ phenotype) was observed in six patients in both the BM and corresponding PB sample, as well as three additional patients with unmatched PB/BM samples. This population was identified using a combination of antibody panels 2 and 4 to first selectively gate the proliferating plasma cells in the CD138+ population (distinct subpopulation) and then investigate their MAGE C1 expression. Fig. 1 shows examples of the two distinct populations observed in the BM. No significant MAGE C1 expression was observed in any of the donor or PB samples using this protocol, confirming a link to the MM diseased state.

Bottom Line: Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls.Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1.MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/-/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples.

View Article: PubMed Central - PubMed

Affiliation: Division of Haematology, University of Cape Town, Cape Town, South Africa.

ABSTRACT
The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/-/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34+/MAGE C1+ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells.

No MeSH data available.


Related in: MedlinePlus