VGH Immunology pioneers NGS HLA typing for Canada: From Serology to Sequencing.

On September 1st 2015, The Vancouver General Hospital Clinical Immunology team and members of UBC Department of Pathology and Laboratory Medicine led by Dr. Paul Keown, announced the evaluation results of a high priority, innovative cross-Canada effort to translate new technologies for HLA typing into the clinical setting. This groundbreaking Canadian effort will offer a new level of diagnostic precision for transplant patients and those with immune disorders, and an important opportunity for improvement of patient care and long-term outcome.

Next Generation Sequencing (NGS) offers enormous potential to enhance the field of HLA typing, through precise determination of nucleotide sequences across the full length of the human genes, phase resolution to minimize ambiguities, multiplexing for scalable analysis, and high-throughput via laboratory automation. The technique reveals entire gene sequences with ultra-high base resolution and can resolve phase ambiguities for multiple samples in a single assay. Implementation of NGS is challenging in clinical diagnostic settings These challenges include adequate computing support and data storage, computational analysis and bioinformatics, and the limitations of reference data in current repositories. However the definite advantages and new diagnostic possibilities make the switch to the technology inevitable.

The Vancouver General Hospital Clinical Immunology Department, in conjunction with the University Of British Columbia Department Of Pathology, held a special 2-day Advances in Transplantation Immunology symposium to address these issues in preparation for the advent of this new technology. Plenary speakers included: i) Dr. Henry Erlich, (Roche Discover Research and Human Genetics), a pioneer of the polymerase chain reaction (PCR) and it’s application in genetic analysis, which has been instrumental in the development of DNA-based HLA typing; ii) Dr. Martin Maiers, (Director of Bioinformatics at the National Marroa Donor program, Minnesota), who is at the forefront of developing systems management of large databases of genetic data relating to the genes of the immune system for application in clinical research and translation into clinical practice.

 

Speakers from the Canadian Advances in Transplantation Immunology Symposium, September 1st 2015. From left; Dr. Duquesnoy, Dr. Wagner, Dr. Minos, Dr. Sherwood, Dr.?, Dr. ?, Dr.?

 

As modern organ transplantation continues to evolve, the Canadian Immunology Network was established to advance transplantation and immunology. It will serve as a source of standardized protocols for human immunology research/clinical tests to facilitate access to material and technologies and to expedite regulatory processes. The network will also lead the development of activities of knowledge dissemination in human immunology at National and International levels and to set up the future for the field to move into. With this in mind, the VGH Clinical Immunology team put forward an innovative strategy on two fronts; standardizing HLA typing across the country and implementing Next Generation Sequencing in the transplantation field.

Traditional methods of using serological typing for HLA has been in use for over 40 years, however there are some drawbacks to serology. The need for viable cells is problematic if the blood has been drawn from deceased donors, as they can’t be stored long-term and may have grossly impaired viability and reduced expression of antigens on the cell surface. Serology is also lacks specificity; for example, there are >300 alleles at the DRB1 locus but only 17 distinct serological specificities or serotypes. In addition, unlike serological typing reagents, the primers/probes and reagents used in PCR-based HLA typing can be produced as standardized reagents. The transition from serology to DNA typing began with PCR-based sequence-specific oligonucleotide/sequence-specific primer (SSO/SSP) technologies. The move was inevitable when published comparative studies of DNA and serological typing, showed that grafts matched using DNA methods survive better than serologically matched grafts (Erlich et al. Immunity. Vol 14, issue 4, p347-356. April 2001). The next logical evolution in the field of HLA DNA typing was to move into Next Generation Sequencing (NGS). NGS is a truly revolutionary technology, allowing accurate, single nucleotide resolution of the amplified locus. This level of high-definition resolution will reveal where and how alleles differ, allowing the analysis of the role of specific polymorphic amino acid residues in peptide binding and presentation as well as in disease association and clinical transplant outcomes.

With the collaborative assistance of multiple industrial partners, our studies show that NGS is fully feasible for routine use and offers precise, ultra-high resolution, complete sequence, cost-efficient high-throughput HLA DNA typing which provides informative data and improved HLA matching for medical research, transplantation medicine, and HLA-related disease diagnosis. Matching solid organ and bone marrow donors to recipients prior to transplant is usually done by analysis of four to six loci (HLA-A, B, C, DR, DQ and DP) but with the accessibility of the new technologies, we are able to provide all 11 loci for the same time-line and prices as more traditional, low/intermediate-resolution DNA-typing tests.

 

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NGS will also open the door to investigating issues of tolerance in transplantation. The best theoretical matches are not always tolerated, and we do no currently understand why some mismatches are well tolerated and others are not. This is an important problem to understand, as Graft vs Host disease (GVHD) is typically severe. The amount of data generated by NGS will make it possible to undertake large scale studies to address this issue. Since the amount of information obtained through NGS will significantly expand our HLA databases, new data management systems will be needed. How the data is stored, accessed and utilized is an important step in the pipeline to clinical transition.

The 2-day symposium also provided an opportunity to discuss the clinical implications of new methods for transplantation matching and management.  The plenary speakers included; Dr. Rene Duquesnoy (Professor Emeritus, University of Pittsburgh), discoverer of the MB system (now called HLA-DQ) and Dr. Anat Tambur (Director of the Transplant Immunology Laboratory at the Comprehensive Transplant Center, Northwestern University), who researches risk stratification of patients. Her recent work has focused on HLA-DQ antigen antibody interactions and the newly emerging concept of HLA epitopes.

Dr. Duquesnoy’s ground-breaking research into epitopes recognized by HLA antibodies and the determination of epitope-based mismatch acceptability for sensitized patients considered for organ transplantation has transformed our understanding of HLA antigenicity. HLA epitope based matching offers new opportunities to tackle the problem of antibody-mediated rejection in solid organ transplantation. Epitope-based interpretations of antibody reactivity patterns with HLA panels in different assays permit more precise assessment of HLA mismatch acceptability for sensitized patients. In addition, epitope-based determinations of HLA mismatch permissibility offer new strategies of reducing antibody responses in non-sensitized patients. As this is a new area of study, there are still many questions that need to be addressed, but the VGH Clinical Immunology laboratory has committed efforts to develop and advance epitope- based acceptability for transplant patients. 

The Canadian Immunology Network has been integral in setting up the HLA and transplant immunology field for the foreseeable future and, in doing so, has placed Canada at the forefront of the transplantation arena.

The team is always open to new collaborative projects. Please feel free to contact Dr. Sherwood at Karen.sherwood@vch.ca for more information.