Utility of a Reverse Phase Protein Array to Evaluate Multiple Biomarkers in Diffuse Large B-Cell Lymphoma

Abstract

Purpose: Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is a heterogeneous lymphoma with different clinical manifestations and molecular alterations, and several markers are currently being measured routinely for its diagnosis, subtyping, or prognostication by immunohistochemistry (IHC). Here, the utility of a reverse-phase-protein-array (RPPA) as a novel supportive tool to measure multiple biomarkers for DLBCL diagnosis is validated.

Experimental design: The expression of seven markers (CD5, CD10, BCL2, BCL6, MUM1, Ki-67, and C-MYC) is analyzed by RPPA and IHC using 37 DLBCL tissues, and the correlation between the two methods is determined. To normalize tumor content ratio in the tissues, the raw RPPA values of each marker are adjusted by that of CD20 or PAX-5.

Results: The CD20-adjusted data for CD5, MUM1, BCL2, Ki-67, and C-MYC has better correlation with IHC results than PAX-5-adjusted data. Receiver operating characteristic (ROC) analysis reveals that CD5, MUM1, BCL2, and C-MYC exhibit a better sensitivity and specificity >0.750. Furthermore, the CD20-adjusted C-MYC value strongly correlates with that of IHC, and has a particularly high specificity (0.882).

Conclusions and clinical relevance: Although further investigation using a large number of DLBCL specimens needs to be conducted, these results suggest that RPPA could be applicable as a supportive tool for determining lymphoma prognosis.

Keywords: C-MYC; diffuse large B-cell lymphoma (DLBCL); immunohistochemistry (IHC); lymphoma prognosis; reverse-phase-protein-array (RPPA).

Figure 1

Validation of monoclonal antibodies (mAbs) by western blot analysis. Total cell lysates (15 µg per lane) prepared from ten cell lines were analyzed by western blot using mAbs against nine biomarkers for DLBCL.

Figure 2

Immunohistochemistry analysis of each marker in DLBCL cells. A–G) DLBCL cells were positive for CD5 (A), CD10 (B), BCL6 (C), MUM1 (D), BCL2 (E), Ki‐67 (F), and C‐MYC (G). H) All cases showed diffuse positive staining for CD20. I) Most cases showed diffuse positive for PAX5, but some cases showed heterogeneous and/or weak PAX5 expression.

Figure 3

a) Correlation between the results of reverse phase protein array (RPPA) and immunohistochemistry (IHC) for each marker. A) For CD5, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. B) For CD10, the correlation coefficient with IHC data was highest in the raw data and lowest in CD20‐adjusted data. C) For BCL6, the correlation coefficient with IHC data was highest in the raw data and lowest in CD20‐adjusted data. D) For MUM1, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. E) For BCL2, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in the raw data. F) For Ki‐67, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in the raw data. G) For C‐MYC, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. R = correlation coefficient. b) Receiver operating characteristic (ROC) analysis of the signal intensities obtained by reverse phase protein array (RPPA) analysis. A) ROC curves of the raw and CD20‐adjusted RPPA data for CD5. B) ROC curves of the raw and CD20‐adjusted RPPA data for CD10. C) ROC curves of the raw and CD20‐adjusted RPPA data for BCL6. D) ROC curves of the raw and CD20‐adjusted RPPA data for MUM1. E) ROC curves of the raw and CD20‐adjusted RPPA data for BCL2. F) ROC curves of the raw and CD20‐adjusted RPPA data for Ki‐67. G) ROC curves of the raw and CD20‐adjusted RPPA data for C‐MYC.

Figure 3

a) Correlation between the results of reverse phase protein array (RPPA) and immunohistochemistry (IHC) for each marker. A) For CD5, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. B) For CD10, the correlation coefficient with IHC data was highest in the raw data and lowest in CD20‐adjusted data. C) For BCL6, the correlation coefficient with IHC data was highest in the raw data and lowest in CD20‐adjusted data. D) For MUM1, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. E) For BCL2, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in the raw data. F) For Ki‐67, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in the raw data. G) For C‐MYC, the correlation coefficient with IHC data was highest in CD20‐adjusted data and lowest in PAX5‐adjusted data. R = correlation coefficient. b) Receiver operating characteristic (ROC) analysis of the signal intensities obtained by reverse phase protein array (RPPA) analysis. A) ROC curves of the raw and CD20‐adjusted RPPA data for CD5. B) ROC curves of the raw and CD20‐adjusted RPPA data for CD10. C) ROC curves of the raw and CD20‐adjusted RPPA data for BCL6. D) ROC curves of the raw and CD20‐adjusted RPPA data for MUM1. E) ROC curves of the raw and CD20‐adjusted RPPA data for BCL2. F) ROC curves of the raw and CD20‐adjusted RPPA data for Ki‐67. G) ROC curves of the raw and CD20‐adjusted RPPA data for C‐MYC.