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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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Immunohistochemistry (IHC) and flow cytometry (FCM) analysis of de novo diffuse large B-cell lymphoma (DLBCL) patients with the CD20 IHC(+)/FCM(−) phenotype. Representative data for four patients are indicated. (a) IHC analysis using anti-CD79a and L26 (anti-CD20) antibody. All those patients were diagnosed as CD79a(+) and CD20(+) de novo DLBCL. (b) FCM analysis of patients showing the CD20 IHC(+)/FCM(−) phenotype. B-cell lymphoma cells were confirmed by gating of SSC, FSC or CD45 expression levels, as well as the CD19-positive phenotype. CD20 expression in those cells was significantly low with FCM analysis. FSC, forward scatter; HE, hematoxylin–eosin staining; Ig, immunoglobulin; L26, anti-CD20 antibody for IHC; Pt #, patient number; SSC; side scatter. Original magnifications (a); ×200 (Olympus BX51TF microscope, Olympus, Tokyo, Japan, and Nikon DS-Fi1 camera, Nikon, Tokyo, Japan).
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fig01: Immunohistochemistry (IHC) and flow cytometry (FCM) analysis of de novo diffuse large B-cell lymphoma (DLBCL) patients with the CD20 IHC(+)/FCM(−) phenotype. Representative data for four patients are indicated. (a) IHC analysis using anti-CD79a and L26 (anti-CD20) antibody. All those patients were diagnosed as CD79a(+) and CD20(+) de novo DLBCL. (b) FCM analysis of patients showing the CD20 IHC(+)/FCM(−) phenotype. B-cell lymphoma cells were confirmed by gating of SSC, FSC or CD45 expression levels, as well as the CD19-positive phenotype. CD20 expression in those cells was significantly low with FCM analysis. FSC, forward scatter; HE, hematoxylin–eosin staining; Ig, immunoglobulin; L26, anti-CD20 antibody for IHC; Pt #, patient number; SSC; side scatter. Original magnifications (a); ×200 (Olympus BX51TF microscope, Olympus, Tokyo, Japan, and Nikon DS-Fi1 camera, Nikon, Tokyo, Japan).

Mentions: Primary or cryopreserved lymphoma tissues showing the CD20 IHC(+)/FCM(−) phenotype obtained in Nagoya University Hospital (n = 8) and the affiliated hospitals (n = 4) were used for further analyses (Table2). Representatives of this phenotype are shown in Figure1(a) (IHC) and 1(b) (FCM). For IHC analysis, B cells were confirmed with anti-CD79a antibody, which recognizes a B-cell receptor component. CD20 protein expression was also confirmed in CD79a-positive B cells (Fig.1a). For FCM analysis, lymphoma cells were gated by side scatter and forward scatter or the CD45 expression level, and CD19-positive B-cell lymphoma populations were confirmed (Fig.1b). However, CD20 expression was not confirmed in these cell populations. B-cell light chain restriction was also confirmed with FCM, and 4 out of 12 cases (33.3%) expressed neither kappa-light chains nor lambda-light chains (Fig.1b and Table2). Interestingly, seven out of eight patients who expressed either the kappa or lambda chain expressed the kappa chain (87.5% of light chain-expressing patients). This percentage in CD20 IHC(+)/FCM(−) patients was higher tendency than that in CD20 IHC(+)/FCM(+) patients (nine out of 20 patients in Table1 [45.0%]).


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)

Immunohistochemistry (IHC) and flow cytometry (FCM) analysis of de novo diffuse large B-cell lymphoma (DLBCL) patients with the CD20 IHC(+)/FCM(−) phenotype. Representative data for four patients are indicated. (a) IHC analysis using anti-CD79a and L26 (anti-CD20) antibody. All those patients were diagnosed as CD79a(+) and CD20(+) de novo DLBCL. (b) FCM analysis of patients showing the CD20 IHC(+)/FCM(−) phenotype. B-cell lymphoma cells were confirmed by gating of SSC, FSC or CD45 expression levels, as well as the CD19-positive phenotype. CD20 expression in those cells was significantly low with FCM analysis. FSC, forward scatter; HE, hematoxylin–eosin staining; Ig, immunoglobulin; L26, anti-CD20 antibody for IHC; Pt #, patient number; SSC; side scatter. Original magnifications (a); ×200 (Olympus BX51TF microscope, Olympus, Tokyo, Japan, and Nikon DS-Fi1 camera, Nikon, Tokyo, Japan).
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Related In: Results  -  Collection

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fig01: Immunohistochemistry (IHC) and flow cytometry (FCM) analysis of de novo diffuse large B-cell lymphoma (DLBCL) patients with the CD20 IHC(+)/FCM(−) phenotype. Representative data for four patients are indicated. (a) IHC analysis using anti-CD79a and L26 (anti-CD20) antibody. All those patients were diagnosed as CD79a(+) and CD20(+) de novo DLBCL. (b) FCM analysis of patients showing the CD20 IHC(+)/FCM(−) phenotype. B-cell lymphoma cells were confirmed by gating of SSC, FSC or CD45 expression levels, as well as the CD19-positive phenotype. CD20 expression in those cells was significantly low with FCM analysis. FSC, forward scatter; HE, hematoxylin–eosin staining; Ig, immunoglobulin; L26, anti-CD20 antibody for IHC; Pt #, patient number; SSC; side scatter. Original magnifications (a); ×200 (Olympus BX51TF microscope, Olympus, Tokyo, Japan, and Nikon DS-Fi1 camera, Nikon, Tokyo, Japan).
Mentions: Primary or cryopreserved lymphoma tissues showing the CD20 IHC(+)/FCM(−) phenotype obtained in Nagoya University Hospital (n = 8) and the affiliated hospitals (n = 4) were used for further analyses (Table2). Representatives of this phenotype are shown in Figure1(a) (IHC) and 1(b) (FCM). For IHC analysis, B cells were confirmed with anti-CD79a antibody, which recognizes a B-cell receptor component. CD20 protein expression was also confirmed in CD79a-positive B cells (Fig.1a). For FCM analysis, lymphoma cells were gated by side scatter and forward scatter or the CD45 expression level, and CD19-positive B-cell lymphoma populations were confirmed (Fig.1b). However, CD20 expression was not confirmed in these cell populations. B-cell light chain restriction was also confirmed with FCM, and 4 out of 12 cases (33.3%) expressed neither kappa-light chains nor lambda-light chains (Fig.1b and Table2). Interestingly, seven out of eight patients who expressed either the kappa or lambda chain expressed the kappa chain (87.5% of light chain-expressing patients). This percentage in CD20 IHC(+)/FCM(−) patients was higher tendency than that in CD20 IHC(+)/FCM(+) patients (nine out of 20 patients in Table1 [45.0%]).

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