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De novo diffuse large B-cell lymphoma with a CD20 immunohistochemistry-positive and flow cytometry-negative phenotype: molecular mechanisms and correlation with rituximab sensitivity.

Tokunaga T, Tomita A, Sugimoto K, Shimada K, Iriyama C, Hirose T, Shirahata-Adachi M, Suzuki Y, Mizuno H, Kiyoi H, Asano N, Nakamura S, Kinoshita T, Naoe T - Cancer Sci. (2013)

Bottom Line: Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(-) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed.Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC.FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(-) cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Hematology/Oncology Research, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.

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In vitro CDC activity induced by rituximab. (a) Annexin V-PI staining was performed with/without rituximab and human serum treatment in vitro. In this assay, living, pro-apoptotic, or dead cell populations were separated in a 2-dimensional graph, and the percentage of each group was calculated. The SU-DHL4 cell line was a positive control. The K562 and WILL2 cell lines were negative controls. Representative primary lymphoma cells showing the IHC(+)/FCM(−) phenotype were obtained from patient #8 and utilized in this assay. (b) The relationship between the percent of cell death with rituximab-induced CDC activity and the CD20-B1-MFI value (performed in Fig.3) was plotted in this graph. Primary lymphoma samples showing CD20 IHC(+)/FCM(+) (black circles) and IHC(+)/FCM(−) (white circles) were used. Each circle indicates one lymphoma sample from a corresponding patient. RRBL1 and WILL2 cells are indicated in black diamonds. (c) The same analysis using the rituximab-MFI values is shown. Nonlinear regression curve fitting is indicated as curved lines. (d) Cell death percentages were statistically compared using Turkey's multiple comparison test. Asterisks indicate significant differences.
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fig04: In vitro CDC activity induced by rituximab. (a) Annexin V-PI staining was performed with/without rituximab and human serum treatment in vitro. In this assay, living, pro-apoptotic, or dead cell populations were separated in a 2-dimensional graph, and the percentage of each group was calculated. The SU-DHL4 cell line was a positive control. The K562 and WILL2 cell lines were negative controls. Representative primary lymphoma cells showing the IHC(+)/FCM(−) phenotype were obtained from patient #8 and utilized in this assay. (b) The relationship between the percent of cell death with rituximab-induced CDC activity and the CD20-B1-MFI value (performed in Fig.3) was plotted in this graph. Primary lymphoma samples showing CD20 IHC(+)/FCM(+) (black circles) and IHC(+)/FCM(−) (white circles) were used. Each circle indicates one lymphoma sample from a corresponding patient. RRBL1 and WILL2 cells are indicated in black diamonds. (c) The same analysis using the rituximab-MFI values is shown. Nonlinear regression curve fitting is indicated as curved lines. (d) Cell death percentages were statistically compared using Turkey's multiple comparison test. Asterisks indicate significant differences.

Mentions: We next performed a rituximab-induced in vitro CDC assay using the same primary lymphoma cells and cell lines as in Figure3. Cells were cultured with or without rituximab for 30 min, and the dead cells were calculated by counting Annexin V- and PI- (or DAPI-) positive cells. Representative data are depicted in Figure4(a). Almost 100% of CD20-positive control SU-DHL4 cells were killed by rituximab-induced CDC activity, but CD20-negative K562 and WILL2 cells were not killed under the same conditions. For the CD20 IHC(+)/FCM(−) cells, partial cell death was observed (Fig.4a, #8). Because normal T cells and/or stromal cells were contaminating cell types in this assay when using primary lymphoma cells from lymphoma tissues, normalization to the B-cell population percentage estimated by determining the CD19-positive cell population was required (data not shown). This normalization for the percent of rituximab-induced cell death was performed for all data obtained from primary lymphoma samples. The relationship between CD20 MFI and the percent of cell death by rituximab-induced CDC activity is indicated in Figure4(b) (MFI; B1) and 4(c) (MFI; rituximab). From these data, a positive correlation was confirmed between the CD20 MFI level and the rituximab effectiveness, as reported previously.23,24 Importantly, rituximab was partially effective on CD20 IHC(+)/FCM(−) cells in vitro (cell death%; range 47–81%) compared to IHC(+)/FCM(+) cells (68–100%) (Fig.4b,c). Significantly lower efficacy of rituximab in the CDC assay in IHC(+)/FCM(−) cells compared with IHC(+)/FCM(+) cells was confirmed (P < 0.05) (Fig.4d). CDC activity was not observed in CD20 IHC(−)/FCM(−) RRBL1 and WILL2 cells (black diamonds in Fig.4b,c). These data suggest that rituximab-induced cytotoxicity can be observed with this CDC assay if the CD20 expression is confirmed with rituximab FCM analysis.


De novo diffuse large B-cell lymphoma with a CD20 immunohistochemistry-positive and flow cytometry-negative phenotype: molecular mechanisms and correlation with rituximab sensitivity.

Tokunaga T, Tomita A, Sugimoto K, Shimada K, Iriyama C, Hirose T, Shirahata-Adachi M, Suzuki Y, Mizuno H, Kiyoi H, Asano N, Nakamura S, Kinoshita T, Naoe T - Cancer Sci. (2013)

In vitro CDC activity induced by rituximab. (a) Annexin V-PI staining was performed with/without rituximab and human serum treatment in vitro. In this assay, living, pro-apoptotic, or dead cell populations were separated in a 2-dimensional graph, and the percentage of each group was calculated. The SU-DHL4 cell line was a positive control. The K562 and WILL2 cell lines were negative controls. Representative primary lymphoma cells showing the IHC(+)/FCM(−) phenotype were obtained from patient #8 and utilized in this assay. (b) The relationship between the percent of cell death with rituximab-induced CDC activity and the CD20-B1-MFI value (performed in Fig.3) was plotted in this graph. Primary lymphoma samples showing CD20 IHC(+)/FCM(+) (black circles) and IHC(+)/FCM(−) (white circles) were used. Each circle indicates one lymphoma sample from a corresponding patient. RRBL1 and WILL2 cells are indicated in black diamonds. (c) The same analysis using the rituximab-MFI values is shown. Nonlinear regression curve fitting is indicated as curved lines. (d) Cell death percentages were statistically compared using Turkey's multiple comparison test. Asterisks indicate significant differences.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317883&req=5

fig04: In vitro CDC activity induced by rituximab. (a) Annexin V-PI staining was performed with/without rituximab and human serum treatment in vitro. In this assay, living, pro-apoptotic, or dead cell populations were separated in a 2-dimensional graph, and the percentage of each group was calculated. The SU-DHL4 cell line was a positive control. The K562 and WILL2 cell lines were negative controls. Representative primary lymphoma cells showing the IHC(+)/FCM(−) phenotype were obtained from patient #8 and utilized in this assay. (b) The relationship between the percent of cell death with rituximab-induced CDC activity and the CD20-B1-MFI value (performed in Fig.3) was plotted in this graph. Primary lymphoma samples showing CD20 IHC(+)/FCM(+) (black circles) and IHC(+)/FCM(−) (white circles) were used. Each circle indicates one lymphoma sample from a corresponding patient. RRBL1 and WILL2 cells are indicated in black diamonds. (c) The same analysis using the rituximab-MFI values is shown. Nonlinear regression curve fitting is indicated as curved lines. (d) Cell death percentages were statistically compared using Turkey's multiple comparison test. Asterisks indicate significant differences.
Mentions: We next performed a rituximab-induced in vitro CDC assay using the same primary lymphoma cells and cell lines as in Figure3. Cells were cultured with or without rituximab for 30 min, and the dead cells were calculated by counting Annexin V- and PI- (or DAPI-) positive cells. Representative data are depicted in Figure4(a). Almost 100% of CD20-positive control SU-DHL4 cells were killed by rituximab-induced CDC activity, but CD20-negative K562 and WILL2 cells were not killed under the same conditions. For the CD20 IHC(+)/FCM(−) cells, partial cell death was observed (Fig.4a, #8). Because normal T cells and/or stromal cells were contaminating cell types in this assay when using primary lymphoma cells from lymphoma tissues, normalization to the B-cell population percentage estimated by determining the CD19-positive cell population was required (data not shown). This normalization for the percent of rituximab-induced cell death was performed for all data obtained from primary lymphoma samples. The relationship between CD20 MFI and the percent of cell death by rituximab-induced CDC activity is indicated in Figure4(b) (MFI; B1) and 4(c) (MFI; rituximab). From these data, a positive correlation was confirmed between the CD20 MFI level and the rituximab effectiveness, as reported previously.23,24 Importantly, rituximab was partially effective on CD20 IHC(+)/FCM(−) cells in vitro (cell death%; range 47–81%) compared to IHC(+)/FCM(+) cells (68–100%) (Fig.4b,c). Significantly lower efficacy of rituximab in the CDC assay in IHC(+)/FCM(−) cells compared with IHC(+)/FCM(+) cells was confirmed (P < 0.05) (Fig.4d). CDC activity was not observed in CD20 IHC(−)/FCM(−) RRBL1 and WILL2 cells (black diamonds in Fig.4b,c). These data suggest that rituximab-induced cytotoxicity can be observed with this CDC assay if the CD20 expression is confirmed with rituximab FCM analysis.

Bottom Line: Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(-) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed.Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC.FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(-) cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Hematology/Oncology Research, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus