Abstract B69: Development of a screening strategy for detecting rituximab-resistant mutations and characterization of mechanisms for acquiring resistant phenotype.
Authors: Mishima Yuji , Terui Yasuhito , Mishima Yuko Matsumoto , Mikuniya Mariko , Hatake Kiyohiko 
Published: June 2012

Abstract
Background: Previously we reported that gene mutations of CD20 were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Most of the cases that had these mutations were diagnosed as CD20 negative by immunohistochemical analysis using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. To make this clear, we determined the binding site of L26 antibody on CD20 protein. In addition, we developed novel antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations.
Methods: To determine the binding site of L26 antibody on CD20, we established six sub-lines that expressed a various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. These cell lines were used for epitope-mapping for L26 antibody. To detect comprehensive CD20 molecules including CD20 having mutation in C-terminal region, we developed antibodies that recognize near the amino terminal of CD20 molecules. Using these antibodies, we re-screened the specimens of CD20 negative cases determined by L26-based immunohistochemistry.
Results: The epitope analysis revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. This result showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. So we screened previously diagnosed specimens and found several cases that included the cells stained by the N-terminal antibody but not by L26. Genetic analysis revealed that these cells had a mutation in the coding region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells having mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids after the amino acid position 250 with a slight early termination. Interestingly, although mutant CD20 molecule expressed adjacent to the plasma membrane, rituximab could not bind to the cells. DNA sequence analysis about genome and mRNA of CD20 gene indicated that the lymphoma cells of this patient had one mutated allele among two CD20 alleles.
Discussions: The C-terminal region of CD20 undertakes a critical role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of C-terminal region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. We propose here that finding a mutation by screening using antibodies of two kinds of epitope is useful in detection of irreversible resistant mutation for molecular targeted drug including rituximab.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B69.