Characterization of Mechanisms for Acquiring Resistant Phenotype Using a Novel Screening Strategy for Detecting Rituximab-Resistant Mutations

Abstract Abstract 1550 Background: We previously reported that mutations of CD20 gene were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be involved in relapse/resistance after rituximab containing therapy. Most of the patients that had mutation in the C-terminal leagion were diagnosed as CD20 negative by immunohistochemistry using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So at first we determined the binding site of L26 antibody on CD20 protein. Then we developed novel diagnostic antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods: To determine the epitope of L26 antibody, we established six sub-lines expressing various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. Then we carried out epitope-mapping using these cell lines. To detect comprehensive CD20 molecules including that having mutation in C-terminal region, we developed antibodies that recognize near the amino terminus of CD20 molecules (CD20N antibody). CD20N antibody is the only monoclonal antibody that recognizes N-terminal region of CD20 so far. Using these antibodies, we screened the specimens of the cases diagnosed as CD20 negative determined by L26-based immunohistochemistry. Results: The epitope-mapping revealed that L26 recognizes near the C-terminus of CD20. This suggested that most of CD20 molecules with the C-terminal deletion mutation or frame-shift mutation could not be recognized by L26. Then we screened previously diagnosed specimens and found several cases that having the cells stained by our novel antibody but not by L26. Genetic analysis revealed that all these cells had a mutation in the C-terminal cytoplasmic region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells with 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 with premature stop at the amino acid position 250. Interestingly, mutant CD20 molecule expressed adjacent to the plasma membrane, but rituximab could not bind to these cells. DNA sequencing study about genome and mRNA of CD20 gene suggested that the lymphoma cells of this patient had one normal and one mutated CD20 allele. Discussions: The C-terminal region of CD20 may undertake a pivotal role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of CD20 in C-terminal cytoplasmic region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. The resistance caused by gene mutation thought to be irreversible. And it should be discriminated from transient downregulation of antigen expression. We propose here that immunohistochemical screening using CD20N antibody is very rapid and effective screening stategy that find out irreversible rituximab resistant cases. Disclosures: No relevant conflicts of interest to declare.

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Anti-N-Terminal CD20 Monoclonal Antibody and L26 Dual Staining Identifies An Irreversible Resistant Mutant Genes

Blood ◽

10.1182/blood.v116.21.4145.4145 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4145-4145

Author(s):

Yuji Mishima ◽

Yasuhito Terui ◽

Yuko Mishima ◽

Kiyohiko Hatake

Keyword(s):

Monoclonal Antibody ◽

Cell Surface ◽

Binding Site ◽

Immunohistochemical Analysis ◽

Surface Expression ◽

Terminal Deletion ◽

Cell Surface Expression ◽

Clinical Specimens ◽

Deletion Mutations ◽

Cytoplasmic Region

Abstract Abstract 4145 [Introduction] Recently, we reported that gene mutations of CD20 were involved in resistance to rituximab therapy, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Many of these cases were diagnosed as CD20 negative by the immunohistochemical analysis using the L26 monoclonal antibody used routinely in most clinical laboratories. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So, we could not distinguish whether protein expression of CD20 extremely decreased or whether the epitope of the antibody was lost by these mutations. To make this clear, we determined the binding site of L26 antibody on CD20 protein in the present study. In addition, we developed new antibodies that recognize amino acid sequence close to the amino terminal of CD20 molecule. Then we investigated clinical specimens with these antibodies together with L26 to elucidate characteristics of CD20 molecules having C-terminal mutations. [Methods] To determine the binding site of L26 antibody on CD20, we made a series of constructs of the CD20 molecules with deletion mutations in the C-terminal cytoplasmic domain and introduced them into retrovirus vectors. A CD20 negative multiple myeloma cell line, KMS12PE cells were then transformed, and we established six kinds of sub-lines with the various C-terminal deletion mutations of CD20 and used them for epitope-mapping. On the other hand, we screened the CD20 gene sequence of the clinical specimen of rituximab-resistant patients and identified several cases with the mutation in the C-terminal cytoplasm region. The immunochemistry using L26 and newly developed antibodies, as well as membrane expression of CD20 molecules using the rituximab were analyzed. [Results] The epitope analysis of L26 antibody using a series of CD20 deletion mutations revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. These results showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. The immunohistochemical analysis performed for clinical specimens revealed that the cells that were stained by antibodies recognizing N-terminal region of CD20 but not by L26 were indeed included in some rituximab-resistant cases. DNA sequencing analysis revealed that all these cases had mutated CD20 genes in its C-terminal cytoplasmic region. In addition, a cell-surface expression analysis using flowcytometry demonstrated that the cells having these mutations has reduced cell surface expression of CD20 compared with those of normal CD20. [Discussion] In this study, we determined the recognition site of L26 and demonstrated that L26 couldn't recognize CD20 with the resistant mutations. In contrast, newly developed antibodies against N-terminal region of CD20 could stain even these CD20 molecules. These results suggest that combination use of these antibodies and L26 enables to detect the onset of irreversible rituximab-resistant clones with the CD20 mutations. Disclosures: Hatake: Chugai Pharmaceutical Co., Ltd: Honoraria, Research Funding.

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Novel Germline CEBPA Sequence Variations in Familial AML and Cytogenetically Normal AML.

Blood ◽

10.1182/blood.v120.21.2566.2566 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2566-2566 ◽

Cited By ~ 1

Author(s):

Rupa A. Udani ◽

Mary Parlow ◽

Lihui Yin ◽

Daniel B. Bellissimo

Keyword(s):

Conflicts Of Interest ◽

Terminal Region ◽

Dominant Negative ◽

Negative Regulator ◽

Buccal Swab ◽

Truncated Form ◽

Frame Shift ◽

Sequence Variations ◽

Cebpa Mutations ◽

Cytogenetically Normal Aml

Abstract Abstract 2566 The CCAAT enhancer binding protein alpha encoded by CEBPA gene is a transcription factors involved in myeloid lineage differentiation. Somatic CEBPA mutations are associated with a favorable prognosis in patients with normal to intermediate karyotype and lacking the internal tandem duplication in the fms-like tyrosine kinase -3 gene (FLT3/ITD) or mutation in the nucleophosmin gene (NPM1). N-terminal CEBPA mutations typically result in a frame shift encoding a truncated form of the 42-kD CEBPα protein and potentially increase translation of the alternative 30-kD isoform. The 30-kD isoform functions as a dominant negative regulator of the full-length 42-kD isoform of the CEBPα protein. C-terminal mutations are generally in-frame insertion/deletions in the DNA binding or the leucine zipper domains that cause alteration of the dimerization domain (bZIP). Germline mutations in CEBPA gene are recognized as the major cause of familial acute myeloid leukemia (AML). Familial AML is inherited in an autosomal dominant manner with complete penetrance. In contrast to somatic AML, the age onset of familial AML is earlier ranging from 4 to 39 years. The overall survival of the familial AML patients with germline CEBPA mutation is ∼50%–65% better than ∼34%–50% observed with the somatic CEBPA mutations (FLT3/NPM1 negative). In all of the familial AML pedigrees reported, the mutations were frame-shift insertions/deletions in the N-terminal domain resulting in translation of a truncated form of the CEBPα protein. We have identified four novel germline sequence variants in patients with history of familial AML and unknown karyotypes (cases 1–4). The patients' age ranges from 3 to 32 years. Of the four novel variants, one variant was identified in the C-terminal region of the gene. This is the first reported C-terminal variant detected in the proband of a familial AML pedigree. The variant is out of frame insertion/deletion of ∼401 bp in the C-terminal region of the CEBPA gene (there is a deletion ‘∼171bp then an insertion of 401bp). Another novel C-terminal variation was identified in a patient with cytogenetically normal AML undergoing testing for somatically acquired CEBPA mutations (case 5). The detection of this C-terminal variation in a buccal swab sample confirmed the germline origin of the variation. In two additional cases (cases 6 and 7), we observed that one of the two sequence variations identified was no longer present after treatment, suggesting the remaining variation was germline in origin. Our results demonstrate that germline C-terminal CEBPA mutations can cause familial AML and that germline CEBPA mutations may be identified in cytogenetically normal AML patients. The possibility of germline variants should be considered in AML patients since other family members, who may be possible transplant donors for the patient, may have also inherited the same germline variant. Case AML Type Sequence variation CEBPA gene region 1 Familial AML c.142delG; p.Ala48fs N terminal 2 c.168C>A; p.Cys56* N terminal 3 c.175G>T; p.Glu59* N terminal 4 c.643_814delins401; p.Thr216fs C terminal 5 Somatic AML c.1073delC; p.Ala358fs C terminal 6 c.68_78del; p.Pro23fs N terminal 7 c. 938T>G; p. Val328G C terminal Disclosures: No relevant conflicts of interest to declare.

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