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Pyrosequencing for Classification of Human FcγRIIIA Allotypes: A Comparison with PCR-Based Techniques

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Abstract

Background

Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP.

Objective

The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes.

Methods

We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches.

Results

The results of our study showed 100 % accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes.

Conclusion

Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

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Acknowledgments

No sources of funding were used to conduct this study or prepare this manuscript. K. Matlawska-Wasowska, J.M. Gale, C.K. Nickl, P. Khalili, B. Shirley, B.S. Wilson, M.A. Vasef and S.S. Winter have no conflicts of interest that are directly relevant to the content of this study. K.M.W. designed and performed the research, wrote and reviewed the manuscript and supervised the project. J.M.G. developed the pyrosequencing protocol, performed the research, wrote and reviewed the manuscript. C.K.N. designed and performed qPCR. P.K. and B.S. were involved in performing pyrosequencing and qPCR, respectively. M.A.V. and B.S.W. participated in designing the research and reviewed the manuscript. S.S.W. originated and supervised the project, wrote and reviewed the manuscript.

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Authors and Affiliations

  1. Department of Pediatrics, Division of Hematology and Oncology, MSC 10 5590, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA

    Ksenia Matlawska-Wasowska, Christian K. Nickl, Brian Shirley & Stuart S. Winter

  2. TriCore Reference Laboratories, Albuquerque, NM, USA

    James M. Gale & Mohammad A. Vasef

  3. Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Parisa Khalili, Bridget S. Wilson & Mohammad A. Vasef

Authors
  1. Ksenia Matlawska-Wasowska
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  2. James M. Gale
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  8. Stuart S. Winter
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Corresponding author

Correspondence to Ksenia Matlawska-Wasowska.

Additional information

K. Matlawska-Wasowska, J. M. Gale contributed equally.

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Matlawska-Wasowska, K., Gale, J.M., Nickl, C.K. et al. Pyrosequencing for Classification of Human FcγRIIIA Allotypes: A Comparison with PCR-Based Techniques. Mol Diagn Ther 18, 665–673 (2014). https://doi.org/10.1007/s40291-014-0120-5

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  • DOI: https://doi.org/10.1007/s40291-014-0120-5

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