J Pathol Transl Med.  2016 Mar;50(2):96-103. 10.4132/jptm.2016.01.12.

Prognostic Implication of Semi-quantitative Immunohistochemical Assessment of CD20 Expression in Diffuse Large B-Cell Lymphoma

• Choi CH
• Park YH
• Lim JH
• Choi SJ
• Kim L
• Park IS
• Han JY
• Kim JM
• Chu YC

• KMID: 2211376
• DOI: http://doi.org/10.4132/jptm.2016.01.12

Abstract

BACKGROUND
Immunohistochemical demonstration of CD20 in diffuse large B-cell lymphoma (DLBCL) is prerequisite not only for the diagnosis but also for assigning patients to rituximab-containing chemotherapy. However, little is known about the impact of abundance of CD20 expression assessed by immunohistochemistry on the clinical outcome of DLBCL. We performed a semi-quantitative immunohistochemical analysis of CD20 expression in DLBCL to examine the prognostic implication of the level of CD20 expression.
METHODS
Pre-treatment diagnostic tissue samples from 48 DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen were represented in a tissue microarray and immunostained for CD20. The relative abundance of CD20 expression was semi-quantitatively scored using a web-based ImmunoMembrane plug-in. Receiver operating characteristic curve analysis was used to determine a prognostically relevant cut-off score in order to dichotomize the patients into CD20-high versus CD20-low groups.
RESULTS
The levels of CD20 expression were heterogeneous among the patients, with a wide and linear distribution of scores. Patients in CD20-low group showed significantly poor clinical outcome.
CONCLUSIONS
The levels of CD20 expression in DLBCL are heterogeneous among the patients with DLBCL. A subgroup of the patients with CD20 expression levels below the cut-off score showed poor clinical outcome.

Keyword

Antigen, CD20; Lymphoma, large B-cell; Immunohistochemistry; Tissue array analysis

MeSH Terms

Antigens, CD20
B-Lymphocytes*
Cyclophosphamide
Diagnosis
Doxorubicin
Drug Therapy
Humans
Immunohistochemistry
Lymphoma, B-Cell*
Prednisone
ROC Curve
Tissue Array Analysis
Vincristine
Rituximab
Antigens, CD20
Cyclophosphamide
Doxorubicin
Prednisone
Vincristine

Figure

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  Fig. 1. Visual heterogeneity in the intensity of CD20 staining in large B-cell lymphoma tumor tissues represented in a tissue microarray. The levels of CD20 expression is visually heterogeneous among tumor tissues (A), ranging from weak (B), intermediate (C), to strong (D) intensity of the 3,3’-diaminobenzidine (DAB) signal.

• 

  Fig. 2. Semi-quantitative immunohistochemical scoring of CD20 expression. The images of weak and incomplete (A, upper), weak and complete (B, upper), and strong and complete (C, upper) staining for CD20 are digitally analyzed using the free web-based ImmunoMembrane (IM) plug-in that produces a combined score of 2 (A, lower), 9 (B, lower), and 20 (C, lower) in its pseudo-color image, respectively.

• 

  Fig. 3. Reproducibility of the immunohistochemical scoring. The combined scores of CD20 expression obtained from the first run of semi-quantitative immunohistochemical scoring are reproducible in the second run. Yellow triangle, CD20-low group; blue triangle, CD20-high group; red rhombus, germinal center of the tonsil as control tissue.

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  Fig. 4. Negatively skewed distribution of the combined scores of CD20 expression. The frequency histogram of the scores illustrates a wide and linear range of the semi-quantitative scores with negatively skewed distribution.

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  Fig. 5. High inter- and intra-group variation in the levels of CD20 expression between patients who survived and those died. The mean of combined scores of CD20 expression was significantly lower in patients who died of disease compared to those who survived (Mann-Whitney test, *p = .012).

• 

  Fig. 6. Determination of a cut-off score of CD20 expression level for poor survival outcome. Receiver operating characteristic (ROC) curve analysis was used to determine a statically optimal cut-off score of CD20 expression level for prediction of disease-associated deaths. The cut-off score of 11.75 derived from the ROC curve analysis maximized the sum of sensitivity and specificity (sensitivity, 72.7%; specificity, 70.3%; p = .012). AUC, area under the ROC curve; CI, confidence interval.

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  Fig. 7. Event-free survival (A) and overall survival (B) of the patients with diffuse large B-cell lymphoma according to the relative abundance of CD20 expression.

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