Limits...
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.

Show MeSH

Related in: MedlinePlus

Flow cytometry (FCM) analyses using anti-CD20 B1 antibody and fluorescent-labeled rituximab. (a) FCM analysis using anti-CD20 B1 antibody was performed, and the MFIs of lymphoma cells were measured. RRBL1 and WILL2 cells were utilized as representative CD20 IHC(−)/FCM(−) samples. The P-value is shown, and the asterisk indicates a statistically significant difference. (b) The MFI value using Alexa 488-labeled rituximab was also analyzed in the same lymphoma samples as (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4317883&req=5

fig03: Flow cytometry (FCM) analyses using anti-CD20 B1 antibody and fluorescent-labeled rituximab. (a) FCM analysis using anti-CD20 B1 antibody was performed, and the MFIs of lymphoma cells were measured. RRBL1 and WILL2 cells were utilized as representative CD20 IHC(−)/FCM(−) samples. The P-value is shown, and the asterisk indicates a statistically significant difference. (b) The MFI value using Alexa 488-labeled rituximab was also analyzed in the same lymphoma samples as (a).

Mentions: To confirm the rituximab effectiveness on CD20 IHC(+)/FCM(−) cells, we first performed FCM analysis using fluorescent (Alexa 488)-labeled rituximab in addition to a conventional anti-CD20 antibody B1 (Dako) (Fig3). We used primary B-cell lymphoma cells and cell lines showing the following phenotypes: CD20 IHC(+)/FCM(+) (primary; n = 10, cell lines; n = 3), IHC(+)/FCM(−) (primary; n = 5) and IHC(−)/FCM(−) after using rituximab (cell lines; n = 2). When using the B1 antibody (Fig.3a), the MFI of CD20 IHC(+)/FCM(−) cells was significantly lower than that of IHC(+)/FCM(+) cells (P = 0.03), consistent with the result of FCM analysis using the B9E9 antibody that recognized the B1 epitope of the CD20 protein (Fig.1b). Using the same cell samples, FCM analysis using Alexa 488-labeled rituximab was also performed (Fig.3b). The MFI of CD20 IHC(+)/FCM(−) cells showed a much lower tendency than that of IHC(+)/FCM(+) cells, but the difference was not significant (P = 0.21). Rituximab, as well as B1, did not detect CD20 expression in the IHC(−)/FCM(−) B-cell lines, RRBL1 and WILL2. These data suggest that CD20 protein is faintly expressed on the surface of CD20 IHC(+)/FCM(−) cells and that rituximab can detect CD20 on the cell surface more effectively than the B1 (B9E9) antibody, even when the expression is very faint.


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)

Flow cytometry (FCM) analyses using anti-CD20 B1 antibody and fluorescent-labeled rituximab. (a) FCM analysis using anti-CD20 B1 antibody was performed, and the MFIs of lymphoma cells were measured. RRBL1 and WILL2 cells were utilized as representative CD20 IHC(−)/FCM(−) samples. The P-value is shown, and the asterisk indicates a statistically significant difference. (b) The MFI value using Alexa 488-labeled rituximab was also analyzed in the same lymphoma samples as (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Flow cytometry (FCM) analyses using anti-CD20 B1 antibody and fluorescent-labeled rituximab. (a) FCM analysis using anti-CD20 B1 antibody was performed, and the MFIs of lymphoma cells were measured. RRBL1 and WILL2 cells were utilized as representative CD20 IHC(−)/FCM(−) samples. The P-value is shown, and the asterisk indicates a statistically significant difference. (b) The MFI value using Alexa 488-labeled rituximab was also analyzed in the same lymphoma samples as (a).
Mentions: To confirm the rituximab effectiveness on CD20 IHC(+)/FCM(−) cells, we first performed FCM analysis using fluorescent (Alexa 488)-labeled rituximab in addition to a conventional anti-CD20 antibody B1 (Dako) (Fig3). We used primary B-cell lymphoma cells and cell lines showing the following phenotypes: CD20 IHC(+)/FCM(+) (primary; n = 10, cell lines; n = 3), IHC(+)/FCM(−) (primary; n = 5) and IHC(−)/FCM(−) after using rituximab (cell lines; n = 2). When using the B1 antibody (Fig.3a), the MFI of CD20 IHC(+)/FCM(−) cells was significantly lower than that of IHC(+)/FCM(+) cells (P = 0.03), consistent with the result of FCM analysis using the B9E9 antibody that recognized the B1 epitope of the CD20 protein (Fig.1b). Using the same cell samples, FCM analysis using Alexa 488-labeled rituximab was also performed (Fig.3b). The MFI of CD20 IHC(+)/FCM(−) cells showed a much lower tendency than that of IHC(+)/FCM(+) cells, but the difference was not significant (P = 0.21). Rituximab, as well as B1, did not detect CD20 expression in the IHC(−)/FCM(−) B-cell lines, RRBL1 and WILL2. These data suggest that CD20 protein is faintly expressed on the surface of CD20 IHC(+)/FCM(−) cells and that rituximab can detect CD20 on the cell surface more effectively than the B1 (B9E9) antibody, even when the expression is very faint.

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