Honors & Awards
New Scholar Award in Aging, Ellison Medical Foundation (2011)
Walter V. and Idun Berry Fellowship, Stanford University (2008)
Harry B. Neustein Award, Society of Pediatric Pathology (2008)
Young Investigator Award, Society of Pediatric Pathology (2007)
Rhodes Scholarship, University of Oxford (1992-4)
Neurofibromatosis (NF) Foundation, 1st Prize, Neurofibromatosis Foundation (2002)
Pre-doctoral fellowship, Howard Hughes Medical Institutes (1997)
Frank Knox Fellowship, Harvard University (1994)
Thomas Jefferson Award, St. Catherines College, Oxford (1994)
Governor Generals Medal, highest undergraduate standing, University of Manitoba (1992)
University Gold Medal, highest standing in honors science, University of Manitoba (1992)
Residency:Stanford University School of Medicine (2007) CA
Fellowship:Stanford University School of Medicine (2008) CA
Medical Education:Harvard Medical School (2005) MA
Ph.D., M.I.T., Biology (2004)
B.A., University of Oxford, English Literature (1994)
B.Sc.(Hons), University of Manitoba, Biochemistry (1992)
Board Certification: Clinical Pathology, American Board of Pathology (2009)
Board Certification: Hematopathology, American Board of Pathology (2009)
Current Research and Scholarly Interests
Our goal is to understand the lymphocyte genotype-phenotype relationships in healthy human immunity and in immunological diseases. We apply new technologies and data analysis approaches to this challenge, particularly high-throughput DNA sequencing and single-cell monoclonal antibody generation, in parallel with functional assays. Our initial focus has been defining clonal lineages of B cells and their antigen specificity in clinical samples from healthy individuals undergoing vaccination or responding to infection, as well as in patients with immune-mediated diseases such as food allergy. Other areas of active research are the B cell responses to HIV, immune deficiencies related to human aging, and transplant immunology.
Integrated Whole-Genome Analysis of Hematologic Disorders
We will use new technologies to look at the DNA, RNA, proteins, and metabolites in the disease-containing blood, bone marrow, or tissue and normal cells from the skin. Our goal is to analyze all of the genes in the diseased and normal skin sample. By comparing the results of the diseased sample and normal skin cells and the results of the two types of genetic information (DNA and RNA), we should be able to identify genetic changes that are important for the initiation, progression, or treatment response of that particular disorder.
Independent Studies (6)
- Directed Reading in Pathology
PATH 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Pathology
PATH 280 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Win, Spr)
- Graduate Research
PATH 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
PATH 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
PATH 199 (Aut, Win, Spr, Sum)
- Directed Reading in Pathology
Graduate and Fellowship Programs
B-cell repertoire responses to varicella-zoster vaccination in human identical twins.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (2): 500-505
Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus.
View details for DOI 10.1073/pnas.1415875112
View details for PubMedID 25535378
Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.
The Journal of allergy and clinical immunology
The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance.We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy.B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones.Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase.Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.
View details for DOI 10.1016/j.jaci.2015.05.029
View details for PubMedID 26152318
Predicting Vaccine Responsiveness.
Cell host & microbe
2015; 17 (3): 301-307
Vaccination has saved many lives and prevented needless suffering from disease, but it is not always effective. Immune responses are a highly "personalized" aspect of an individual's biology, as they are subject to germline genetic influences but are embodied in cell populations that continuously sample the environment. Additionally, immunity is shaped by memory of prior infectious diseases and other antigenic exposures. Here, we review examples of recent technical advances and insights into human vaccine responses that are helping to define the features associated with successful vaccination and that may enable a more predictive vaccinology in the future.
View details for DOI 10.1016/j.chom.2015.02.015
View details for PubMedID 25766292
HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria.
Cell host & microbe
2014; 16 (2): 215-226
Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.
View details for DOI 10.1016/j.chom.2014.07.003
View details for PubMedID 25121750
Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.
Cell host & microbe
2014; 16 (1): 105-114
B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
View details for DOI 10.1016/j.chom.2014.05.013
View details for PubMedID 24981332
Effects of Aging, Cytomegalovirus Infection, and EBV Infection on Human B Cell Repertoires
JOURNAL OF IMMUNOLOGY
2014; 192 (2): 603-611
Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.
View details for DOI 10.4049/jimmunol.1301384
View details for Web of Science ID 000329224000006
View details for PubMedID 24337376
An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1.
The Journal of clinical investigation
Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.
View details for DOI 10.1172/JCI73441
View details for PubMedID 24614107
Diversity and clonal selection in the human T-cell repertoire.
Proceedings of the National Academy of Sciences of the United States of America
T-cell receptor (TCR) diversity, a prerequisite for immune system recognition of the universe of foreign antigens, is generated in the first two decades of life in the thymus and then persists to an unknown extent through life via homeostatic proliferation of naïve T cells. We have used next-generation sequencing and nonparametric statistical analysis to estimate a lower bound for the total number of different TCR beta (TCRB) sequences in human repertoires. We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in naïve CD4 and CD8 T-cell repertoires of young adults. Naïve repertoire richness modestly declined two- to fivefold in healthy elderly. Repertoire richness contraction with age was even less pronounced for memory CD4 and CD8 T cells. In contrast, age had a major impact on the inequality of clonal sizes, as estimated by a modified Gini-Simpson index clonality score. In particular, large naïve T-cell clones that were distinct from memory clones were found in the repertoires of elderly individuals, indicating uneven homeostatic proliferation without development of a memory cell phenotype. Our results suggest that a highly diverse repertoire is maintained despite thymic involution; however, peripheral fitness selection of T cells leads to repertoire perturbations that can influence the immune response in the elderly.
View details for DOI 10.1073/pnas.1409155111
View details for PubMedID 25157137
Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies.
Cell host & microbe
2014; 16 (3): 304-13
Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.
View details for DOI 10.1016/j.chom.2014.08.006
View details for PubMedID 25211073
Convergent Antibody Signatures in Human Dengue
CELL HOST & MICROBE
2013; 13 (6): 691-700
Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.
View details for DOI 10.1016/j.chom.2013.05.008
View details for Web of Science ID 000330851000009
Diagnostic Applications of High-Throughput DNA Sequencing
ANNUAL REVIEW OF PATHOLOGY: MECHANISMS OF DISEASE, VOL 8
2013; 8: 381-410
Advances in DNA sequencing technology have allowed comprehensive investigation of the genetics of human beings and human diseases. Insights from sequencing the genomes, exomes, or transcriptomes of healthy and diseased cells in patients are already enabling improved diagnostic classification, prognostication, and therapy selection for many diseases. Understanding the data obtained using new high-throughput DNA sequencing methods, choices made in sequencing strategies, and common challenges in data analysis and genotype-phenotype correlation is essential if pathologists, geneticists, and clinicians are to interpret the growing scientific literature in this area. This review highlights some of the major results and discoveries stemming from high-throughput DNA sequencing research in our understanding of Mendelian genetic disorders, hematologic cancer biology, infectious diseases, the immune system, transplant biology, and prenatal diagnostics. Transition of new DNA sequencing methodologies to the clinical laboratory is under way and is likely to have a major impact on all areas of medicine.
View details for DOI 10.1146/annurev-pathol-020712-164026
View details for Web of Science ID 000318483400015
View details for PubMedID 23121054
- Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing Science Translational Medicine 2009; 1 (12)
Design of a Genomics Curriculum: Competencies for Practicing Pathologists.
Archives of pathology & laboratory medicine
2015; 139 (7): 894-900
Context .- The field of genomics is rapidly impacting medical care across specialties. To help guide test utilization and interpretation, pathologists must be knowledgeable about genomic techniques and their clinical utility. The technology allowing timely generation of genomic data is relatively new to patient care and the clinical laboratory, and therefore, many currently practicing pathologists have been trained without any molecular or genomics exposure. Furthermore, the exposure that current and recent trainees receive in this field remains inconsistent. Objective .- To assess pathologists' learning needs in genomics and to develop a curriculum to address these educational needs. Design .- A working group formed by the College of American Pathologists developed an initial list of genomics competencies (knowledge and skills statements) that a practicing pathologist needs to be successful. Experts in genomics were then surveyed to rate the importance of each competency. These data were used to create a final list of prioritized competencies. A subset of the working group defined subtopics and tasks for each competency. Appropriate delivery methods for the educational material were also proposed. Results .- A final list of 32 genomics competency statements was developed. A prioritized curriculum was created with designated subtopics and tasks associated with each competency. Conclusions .- We present a genomics curriculum designed as a first step toward providing practicing pathologists with the competencies needed to practice successfully.
View details for DOI 10.5858/arpa.2014-0253-CP
View details for PubMedID 26125429
A Balanced Look at the Implications of Genomic (and Other "Omics") Testing for Disease Diagnosis and Clinical Care.
2014; 5 (3): 748-766
The tremendous increase in DNA sequencing capacity arising from the commercialization of "next generation" instruments has opened the door to innumerable routes of investigation in basic and translational medical science. It enables very large data sets to be gathered, whose interpretation and conversion into useful knowledge is only beginning. A challenge for modern healthcare systems and academic medical centers is to apply these new methods for the diagnosis of disease and the management of patient care without unnecessary delay, but also with appropriate evaluation of the quality of data and interpretation, as well as the clinical value of the insights gained. Most critically, the standards applied for evaluating these new laboratory data and ensuring that the results and their significance are clearly communicated to patients and their caregivers should be at least as rigorous as those applied to other kinds of medical tests. Here, we present an overview of conceptual and practical issues to be considered in planning for the integration of genomic methods or, in principle, any other type of "omics" testing into clinical care.
View details for DOI 10.3390/genes5030748
View details for PubMedID 25257203
High-Throughput DNA Sequencing Analysis of Antibody Repertoires.
2014; 2 (5)
New high-throughput DNA sequencing (HTS) technologies developed in the past decade have begun to be applied to the study of the complex gene rearrangements that encode human antibodies. This article first reviews the genetic features of Ig loci and the HTS technologies that have been applied to human repertoire studies, then discusses key choices for experimental design and data analysis in these experiments and the insights gained in immunological and infectious disease studies with the use of these approaches.
View details for DOI 10.1128/microbiolspec.AID-0017-2014
View details for PubMedID 26104353
Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes.
2013; 98 (11): 1689-1696
In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).
View details for DOI 10.3324/haematol.2013.092379
View details for PubMedID 23872309
Human lymphocyte repertoires in ageing.
Current opinion in immunology
2013; 25 (4): 511-515
Deterioration of adaptive immunity with ageing may reflect changes in the repertoire of T cells and B cells available to respond to antigenic challenges, due to altered proportions and absolute numbers of lymphocyte subpopulations as well as changes in the repertoire of antigen receptor genes expressed by these cells. High-throughput DNA sequencing (HTS) now facilitates examination of immunoglobulin and T cell receptor gene rearrangements, and initial studies using these methods to study immune system ageing in humans have demonstrated age-related alterations in the receptor populations within lymphocyte subsets, as well as in repertoires responding to vaccination. Accurate measurement of repertoire diversity remains an experimental challenge. Studies of larger numbers of human subjects, analysis of defined lymphocyte subpopulations including antigen-specific populations, and controlling for factors such as chronic viral infections, will be important for gaining additional understanding of the impact of ageing on human lymphocyte populations.
View details for DOI 10.1016/j.coi.2013.07.007
View details for PubMedID 23992996
Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.
2013; 496 (7446): 469-476
Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.
View details for DOI 10.1038/nature12053
View details for PubMedID 23552890
Selective Immunophenotyping for Diagnosis of B-cell Neoplasms: Immunohistochemistry and Flow Cytometry Strategies and Results
APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY
2013; 21 (2): 116-131
Determining the immunophenotype of hematologic malignancies is now an indispensable part of diagnostic classification, and can help to guide therapy, or to predict clinical outcome. Diagnostic workup should be guided by morphologic findings and evaluate clinically important markers, but ideally should avoid the use of overly broad panels of immunostains that can reveal incidental findings of uncertain significance and give rise to increased costs. Here, we outline our approach to diagnosis of B-cell neoplasms, combining histologic and clinical data with tailored panels of immunophenotyping reagents, in the context of the 2008 World Health Organization classification. We present data from cases seen at our institution from 2004 through 2008 using this approach, to provide a practical reference for findings seen in daily diagnostic practice.
View details for DOI 10.1097/PAI.0b013e31825d550a
View details for Web of Science ID 000315464500004
View details for PubMedID 22820658
Integration of Genomic Medicine into Pathology Residency Training The Stanford Open Curriculum
JOURNAL OF MOLECULAR DIAGNOSTICS
2013; 15 (2): 141-148
Next-generation sequencing methods provide an opportunity for molecular pathology laboratories to perform genomic testing that is far more comprehensive than single-gene analyses. Genome-based test results are expected to develop into an integral component of diagnostic clinical medicine and to provide the basis for individually tailored health care. To achieve these goals, rigorous interpretation of high-quality data must be informed by the medical history and the phenotype of the patient. The discipline of pathology is well positioned to implement genome-based testing and to interpret its results, but new knowledge and skills must be included in the training of pathologists to develop expertise in this area. Pathology residents should be trained in emerging technologies to integrate genomic test results appropriately with more traditional testing, to accelerate clinical studies using genomic data, and to help develop appropriate standards of data quality and evidence-based interpretation of these test results. We have created a genomic pathology curriculum as a first step in helping pathology residents build a foundation for the understanding of genomic medicine and its implications for clinical practice. This curriculum is freely accessible online.
View details for DOI 10.1016/j.jmoldx.2012.11.003
View details for Web of Science ID 000315841600001
View details for PubMedID 23313248
New tools for classification and monitoring of autoimmune diseases
NATURE REVIEWS RHEUMATOLOGY
2012; 8 (6): 317-328
Rheumatologists see patients with a range of autoimmune diseases. Phenotyping these diseases for diagnosis, prognosis and selection of therapies is an ever increasing problem. Advances in multiplexed assay technology at the gene, protein, and cellular level have enabled the identification of 'actionable biomarkers'; that is, biological metrics that can inform clinical practice. Not only will such biomarkers yield insight into the development, remission, and exacerbation of a disease, they will undoubtedly improve diagnostic sensitivity and accuracy of classification, and ultimately guide treatment. This Review provides an introduction to these powerful technologies that could promote the identification of actionable biomarkers, including mass cytometry, protein arrays, and immunoglobulin and T-cell receptor high-throughput sequencing. In our opinion, these technologies should become part of routine clinical practice for the management of autoimmune diseases. The use of analytical tools to deconvolve the data obtained from use of these technologies is also presented here. These analyses are revealing a more comprehensive and interconnected view of the immune system than ever before and should have an important role in directing future treatment approaches for autoimmune diseases.
View details for DOI 10.1038/nrrheum.2012.66
View details for Web of Science ID 000304719600005
View details for PubMedID 22647780
The Inference of Phased Haplotypes for the Immunoglobulin H Chain V Region Gene Loci by Analysis of VDJ Gene Rearrangements
JOURNAL OF IMMUNOLOGY
2012; 188 (3): 1333-1340
The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV?IGHD?IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.
View details for DOI 10.4049/jimmunol.1102097
View details for Web of Science ID 000299690200048
View details for PubMedID 22205028
High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (52): 21194-21199
The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.
View details for DOI 10.1073/pnas.1118357109
View details for Web of Science ID 000298479900065
View details for PubMedID 22160699
Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated
JOURNAL OF EXPERIMENTAL MEDICINE
2011; 208 (11): 2237-2249
The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.
View details for DOI 10.1084/jem.20110363
View details for Web of Science ID 000296537800011
View details for PubMedID 21987658
Determinants of nucleosome organization in primary human cells
2011; 474 (7352): 516-U148
Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.
View details for DOI 10.1038/nature10002
View details for Web of Science ID 000291939700050
View details for PubMedID 21602827
Benchmarking the performance of human antibody gene alignment utilities using a 454 sequence dataset
2010; 26 (24): 3129-3130
Immunoglobulin heavy chain genes are formed by recombination of genes randomly selected from sets of IGHV, IGHD and IGHJ genes. Utilities have been developed to identify genes that contribute to observed VDJ rearrangements, but in the absence of datasets of known rearrangements, the evaluation of these utilities is problematic. We have analyzed thousands of VDJ rearrangements from an individual (S22) whose IGHV, IGHD and IGHJ genotype can be inferred from the dataset. Knowledge of this genotype means that the Stanford_S22 dataset can serve to benchmark the performance of IGH alignment utilities.We evaluated the performance of seven utilities. Failure to partition a sequence into genes present in the S22 genome was considered an error, and error rates for different utilities ranged from 7.1% to 13.7%.Supplementary data includes the S22 genotypes and alignments. The Stanford_S22 dataset and an evaluation tool is available at http://www.emi.unsw.edu.au/~ihmmune/IGHUtilityEval/.
View details for DOI 10.1093/bioinformatics/btq604
View details for Web of Science ID 000284947700019
View details for PubMedID 21036814
Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements
JOURNAL OF IMMUNOLOGY
2010; 184 (12): 6986-6992
Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.
View details for DOI 10.4049/jimmunol.1000445
View details for Web of Science ID 000278516700047
View details for PubMedID 20495067
A Comparison of Two Methods for Screening CEBPA Mutations in Patients with Acute Myeloid Leukemia
JOURNAL OF MOLECULAR DIAGNOSTICS
2009; 11 (4): 319-323
The goal of the study was to compare the performance of a fluorescence-based multiplex PCR fragment analysis to a direct sequencing method for detecting CEBPA mutations in patients with acute myeloid leukemia. Thirty-three samples were selected from a larger study of 107 cases of acute myeloid leukemia by screening for CEBPA mutations by sequence analysis. Of ten identified mutations, six (insertions and deletions) were detected by both sequencing and fragment methods. The fragment analysis method did not detect the remaining four base substitutions because the method cannot detect changes that result in identically sized products. The multiplex PCR fragment length analysis method therefore failed to detect substitution mutations accounting for 40% of total CEBPA mutations in our patient set. Our results indicate that fragment length analysis should not be used in isolation, and that direct sequencing is required to evaluate CEBPA gene mutational status in acute myeloid leukemia. A combination of the two assays may offer some advantages, chiefly in permitting more sensitive detection by fragment length analysis of insertions and deletions.
View details for DOI 10.2353/jmoldx.2009.080121
View details for Web of Science ID 000267562000008
View details for PubMedID 19525338
Features of hemolysis due to Clostridium perfringens infection
INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY
2009; 31 (3): 364-367
Infection by Clostridium perfringens can be an unsuspected cause of hemolysis in emergency room patients. Historically, this condition has been associated with wound contamination and other tissue infections. We report the case of an autistic patient who presented to our emergency department with a distended abdomen and hemolysis of unknown etiology. The patient had no history of recent surgery. Exploration of the abdomen revealed a hepatic abscess. Blood cultures tested culture positive for C. perfringens. We present images demonstrating the salient features of the peripheral blood smear in cases of this uncommon but deadly cause of hemolysis.
View details for DOI 10.1111/j.1751-553X.2007.01018.x
View details for Web of Science ID 000265407400013
View details for PubMedID 18177433
Everything you wanted to know about small RNA but were afraid to ask
2008; 88 (6): 569-578
MicroRNAs are a class of recently discovered small RNA molecules that regulate other genes in the human genome. Studies in human cells and model organisms have begun to reveal the mechanisms of microRNA activity, and the wide range of normal physiological functions they influence. Their alteration in pathologic states from cancer to cardiovascular disease is also increasingly clear. A review of current evidence for the role of these molecules in human health and disease will be helpful to pathologists and medical researchers as the fascinating story of these small regulators continues to unfold.
View details for DOI 10.1038/labinvest.2008.32
View details for Web of Science ID 000256114600001
View details for PubMedID 18427554
Alloimmunization to red blood cell antigens affects clinical outcomes in liver transplant patients
2007; 13 (12): 1654-1661
Transfusion therapy of liver transplant patients remains a challenge. High volumes of intraoperative blood transfusion have been shown to increase the risk of poor graft or patient survival. We conducted a retrospective study of 209 consecutive liver transplant cases at our institution. Only patients receiving their first liver transplant, with no other simultaneous organ transplants, were included. Cox proportional hazard modeling was used to identify clinical variables correlated with postoperative patient mortality. Statistically significant variables for poor patient survival were the number of red blood cell and plasma units transfused, a history of red blood cell alloantibodies, and the immunosuppressive regimen used. History of pregnancy also approached statistical significance but was less robust than the other 3 variables. Our findings suggest that blood transfusion and immune modulation greatly affect the survival of patients after liver transplantation.
View details for DOI 10.1002/It.21241
View details for Web of Science ID 000251574100007
View details for PubMedID 18044783
An intact HDM2 RING-finger domain is required for nuclear exclusion of p53
NATURE CELL BIOLOGY
2000; 2 (9): 563-568
The p53 tumour-suppressor protein is negatively regulated by HDM2. Recent reports indicate that the leucine-rich nuclear-export sequence (NES) of HDM2 enables it to shuttle to the cytoplasm, and that this activity is required for degradation of p53. However, it is unclear whether HDM2 is involved in nuclear export of p53, partly because p53 has itself been shown to contain a functional NES within its tetramerization domain. Here we show that co-expression of HDM2 with green fluorescent protein (GFP)-tagged p53 causes redistribution of p53 from the nucleus to the cytoplasm of the cell. This activity is dependent on binding of p53 to HDM2, and requires an intact p53 NES, but is independent of the HDM2 NES. A mutant of the HDM2 RING-finger domain that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that ubiquitin ligation or other activities of this region of HDM2 may be necessary for its regulation of p53 localization.
View details for Web of Science ID 000089250600006
View details for PubMedID 10980695
The role of CTLA-4 in regulating Th2 differentiation
JOURNAL OF IMMUNOLOGY
1999; 163 (5): 2634-2639
To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.
View details for Web of Science ID 000082125400039
View details for PubMedID 10453003
Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G(1) arrest after gamma-irradiation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (3): 1002-1007
In mammalian cells, activation of certain checkpoint pathways as a result of exposure to genotoxic agents results in cell cycle arrest. The integrity of these arrest pathways is critical to the ability of the cell to repair mutations that otherwise might compromise viability or contribute to deregulation of cellular growth and proliferation. Here we examine the mechanism through which DNA damaging agents result in a G1 arrest that depends on the tumor suppressor p53 and its transcriptional target p21. By using primary cell lines lacking specific cell cycle regulators, we demonstrate that this pathway functions through the growth suppressive properties of the retinoblastoma protein (pRB) tumor suppressor. Specifically, gamma-irradiation inhibits the phosphorylation of pRB at cyclin-dependent kinase 2-specific, but not cyclin-dependent kinase 4-specific, sites in a p21-dependent manner. Most importantly, we show that pRB is a critical component of this DNA damage checkpoint. These data indicate that the p53 --> p21 checkpoint pathway uses the normal cell cycle regulatory machinery to induce the accumulation of the growth suppressive form of pRB and suggest that loss of pRB during the course of tumorigenesis disrupts the function of an important DNA damage checkpoint.
View details for Web of Science ID 000078484100038
View details for PubMedID 9927683
CTLA4Ig prevents lymphoproliferation and fatal multiorgan tissue destruction in CTLA-4-deficient mice
JOURNAL OF IMMUNOLOGY
1997; 158 (11): 5091-5094
Mice lacking CTLA-4 develop a fatal spontaneous lymphoproliferative disease with massive lymphocytic infiltrates and tissue destruction in many organs. CTLA-4-deficient (-/-) splenocytes and lymph node cells proliferate without added stimuli in vitro. We report here that CTLA4Ig treatment of CTLA-4 -/- mice prevents lymphoproliferation and fatal multiorgan tissue damage in vivo and proliferation of CTLA-4 -/- splenocytes and lymph node cells in vitro. Therefore, stimulation via CD28-B7 interactions appears necessary for CTLA-4 -/- T cell proliferation and the production of lymphoproliferative disease in vivo. When CTLA4Ig treatment is terminated, CTLA-4 -/- T cells become activated and lymphoproliferative disease develops. The lack of long term protective effects of CTLA4Ig treatment suggests that CTLA-4 is needed for the induction and or maintenance of tolerance.
View details for Web of Science ID A1997XA06300007
View details for PubMedID 9164923
B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation
1997; 6 (3): 303-313
Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.
View details for Web of Science ID A1997XC61800010
View details for PubMedID 9075931