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A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia.

Managò S, Valente C, Mirabelli P, Circolo D, Basile F, Corda D, De Luca AC - Sci Rep (2016)

Bottom Line: B-ALL diagnosis requires identification and classification of the leukemia cells.In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages.Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, 80131 Naples, Italy.

ABSTRACT
Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

No MeSH data available.


Related in: MedlinePlus

Effect of ATRA treatment on acute lymphocytic leukemia cells.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells treated with 1 μM ATRA for 72 h, and fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red) to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 B-leukemia cells treated as in a, with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Raman spectra of three B-leukemia cell lines recorded without and with ATRA treatment as in. (a) The difference spectra were obtained by subtracting the untreated from the treated spectra (bottom panels). (d) PCA scatter plots comparing the untreated and ATRA-treated B-leukemia cells. (e) Confusion matrix for the classification of the untreated and ATRA-treated B-leukemia cells.
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f4: Effect of ATRA treatment on acute lymphocytic leukemia cells.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells treated with 1 μM ATRA for 72 h, and fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red) to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 B-leukemia cells treated as in a, with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Raman spectra of three B-leukemia cell lines recorded without and with ATRA treatment as in. (a) The difference spectra were obtained by subtracting the untreated from the treated spectra (bottom panels). (d) PCA scatter plots comparing the untreated and ATRA-treated B-leukemia cells. (e) Confusion matrix for the classification of the untreated and ATRA-treated B-leukemia cells.

Mentions: To provide a scenario as close as possible to the clinical setting, we analyzed these particular RS variations under ‘treatment’ with two clinically demonstrated beneficial drugs that are used in maintenance chemotherapy of patients with B-ALL: MTX and 6MP. Low doses of MTX and 6MP that provide patients with peak plasma concentrations range from 0.01 μM to 1 μM41 are used to induce specific B-ALL regression. MTX and 6MP are also specific treatments for patients with B-ALL, as they do not provide benefits for other leukemias, such as for regression of acute promyelocytic leukemia, where the maintenance therapy is primarily ATRA41. These three transformed B-leukemia cell lines were thus treated for 72 h with 1 μM MTX, 1 μM 6MP, or 1 μM ATRA (as the control)41. The cells were then analyzed by immunofluorescence microscopy and Western blotting to determine whether the B-ALL regression obtained in patients under similar conditions is associated with decreased expression of particular B-leukemia antigens. This MTX treatment significantly reduced the plasma membrane staining of CD38 and CD19 in all of these B-leukemia cell lines, along with that of CD10 in the REH and MN60 cells, and of CD20 in the MN60 cells (Fig. 3a versus non-treated control cells shown in Fig. 2a). These immunofluorescence data were supported and confirmed by Western blotting (Fig. 3b). Moreover, reduced intensity of nucleic-acid fluorescence staining was also observed in all three of these MTX-treated B-leukemia cell lines (Fig. 3a versus non-treated control cells shown in Fig. 2a). Similar data for both antigen expression and nucleic acids (stained with Hoechst dye) were obtained with 6MP treatment (data not shown), while no effects were detected with ATRA treatment (Fig. 4a,b). Flow Cytometry analysis confirmed confocal microscopy study regarding the down-regulation of CD19, CD10 and CD38 differentiation antigens upon induction with MTX, while no effects were detected with ATRA treatment, as shown in Supplementary Fig. 3. These data indicate that MTX treatment specifically reverted the B-cell differentiation process for these L2 (RS4;11 and REH cells) and L3 (MN60 cells) subtypes of B-leukemia cells (Fig. 3a,b), while as expected, the ATRA acute promyelocytic leukemia treatment had no effect (Fig. 4a,b). Of note, under these conditions, no drug-mediated apoptotic effects were detected, as indicated by the nucleolus and chromatin staining with the Hoechst 33258 dye (Figs 3a and 4a)55.


A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia.

Managò S, Valente C, Mirabelli P, Circolo D, Basile F, Corda D, De Luca AC - Sci Rep (2016)

Effect of ATRA treatment on acute lymphocytic leukemia cells.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells treated with 1 μM ATRA for 72 h, and fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red) to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 B-leukemia cells treated as in a, with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Raman spectra of three B-leukemia cell lines recorded without and with ATRA treatment as in. (a) The difference spectra were obtained by subtracting the untreated from the treated spectra (bottom panels). (d) PCA scatter plots comparing the untreated and ATRA-treated B-leukemia cells. (e) Confusion matrix for the classification of the untreated and ATRA-treated B-leukemia cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Effect of ATRA treatment on acute lymphocytic leukemia cells.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells treated with 1 μM ATRA for 72 h, and fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red) to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 B-leukemia cells treated as in a, with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Raman spectra of three B-leukemia cell lines recorded without and with ATRA treatment as in. (a) The difference spectra were obtained by subtracting the untreated from the treated spectra (bottom panels). (d) PCA scatter plots comparing the untreated and ATRA-treated B-leukemia cells. (e) Confusion matrix for the classification of the untreated and ATRA-treated B-leukemia cells.
Mentions: To provide a scenario as close as possible to the clinical setting, we analyzed these particular RS variations under ‘treatment’ with two clinically demonstrated beneficial drugs that are used in maintenance chemotherapy of patients with B-ALL: MTX and 6MP. Low doses of MTX and 6MP that provide patients with peak plasma concentrations range from 0.01 μM to 1 μM41 are used to induce specific B-ALL regression. MTX and 6MP are also specific treatments for patients with B-ALL, as they do not provide benefits for other leukemias, such as for regression of acute promyelocytic leukemia, where the maintenance therapy is primarily ATRA41. These three transformed B-leukemia cell lines were thus treated for 72 h with 1 μM MTX, 1 μM 6MP, or 1 μM ATRA (as the control)41. The cells were then analyzed by immunofluorescence microscopy and Western blotting to determine whether the B-ALL regression obtained in patients under similar conditions is associated with decreased expression of particular B-leukemia antigens. This MTX treatment significantly reduced the plasma membrane staining of CD38 and CD19 in all of these B-leukemia cell lines, along with that of CD10 in the REH and MN60 cells, and of CD20 in the MN60 cells (Fig. 3a versus non-treated control cells shown in Fig. 2a). These immunofluorescence data were supported and confirmed by Western blotting (Fig. 3b). Moreover, reduced intensity of nucleic-acid fluorescence staining was also observed in all three of these MTX-treated B-leukemia cell lines (Fig. 3a versus non-treated control cells shown in Fig. 2a). Similar data for both antigen expression and nucleic acids (stained with Hoechst dye) were obtained with 6MP treatment (data not shown), while no effects were detected with ATRA treatment (Fig. 4a,b). Flow Cytometry analysis confirmed confocal microscopy study regarding the down-regulation of CD19, CD10 and CD38 differentiation antigens upon induction with MTX, while no effects were detected with ATRA treatment, as shown in Supplementary Fig. 3. These data indicate that MTX treatment specifically reverted the B-cell differentiation process for these L2 (RS4;11 and REH cells) and L3 (MN60 cells) subtypes of B-leukemia cells (Fig. 3a,b), while as expected, the ATRA acute promyelocytic leukemia treatment had no effect (Fig. 4a,b). Of note, under these conditions, no drug-mediated apoptotic effects were detected, as indicated by the nucleolus and chromatin staining with the Hoechst 33258 dye (Figs 3a and 4a)55.

Bottom Line: B-ALL diagnosis requires identification and classification of the leukemia cells.In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages.Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, 80131 Naples, Italy.

ABSTRACT
Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

No MeSH data available.


Related in: MedlinePlus