John D Pfeifer

Harvard Medical School, Boston, Massachusetts, United States

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Publications (138)514.92 Total impact

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    ABSTRACT: Objectives: To evaluate the extent of human-to-human specimen contamination in clinical next-generation sequencing (NGS) data. Methods: Using haplotype analysis to detect specimen admixture, with orthogonal validation by short tandem repeat analysis, we determined the rate of clinically significant (>5%) DNA contamination in clinical NGS data from 296 consecutive cases. Haplotype analysis was performed using read haplotypes at common, closely spaced single-nucleotide polymorphisms in low linkage disequilibrium in the population, which were present in regions targeted by the clinical assay. Percent admixture was estimated based on frequencies of the read haplotypes at loci that showed evidence for contamination. Results: We identified nine (3%) cases with at least 5% DNA admixture. Three cases were bone marrow transplant patients known to be chimeric. Six admixed cases were incidents of contamination, and the rate of contamination was strongly correlated with DNA yield from the tissue specimen. Conclusions: Human-human specimen contamination occurs in clinical NGS testing. Tools for detecting contamination in NGS sequence data should be integrated into clinical bioinformatics pipelines, especially as laboratories trend toward using smaller amounts of input DNA and reporting lower frequency variants. This study provides one estimate of the rate of clinically significant human-human specimen contamination in clinical NGS testing.
    American Journal of Clinical Pathology 09/2015; 144(4):667-74. DOI:10.1309/AJCPR88WDJJLDMBN · 2.51 Impact Factor
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    ABSTRACT: Context .- We define the scope and needs within the new discipline of computational pathology, a discipline critical to the future of both the practice of pathology and, more broadly, medical practice in general. Objective .- To define the scope and needs of computational pathology. Data Sources .- A meeting was convened in Boston, Massachusetts, in July 2014 prior to the annual Association of Pathology Chairs meeting, and it was attended by a variety of pathologists, including individuals highly invested in pathology informatics as well as chairs of pathology departments. Conclusions .- The meeting made recommendations to promote computational pathology, including clearly defining the field and articulating its value propositions; asserting that the value propositions for health care systems must include means to incorporate robust computational approaches to implement data-driven methods that aid in guiding individual and population health care; leveraging computational pathology as a center for data interpretation in modern health care systems; stating that realizing the value proposition will require working with institutional administrations, other departments, and pathology colleagues; declaring that a robust pipeline should be fostered that trains and develops future computational pathologists, for those with both pathology and nonpathology backgrounds; and deciding that computational pathology should serve as a hub for data-related research in health care systems. The dissemination of these recommendations to pathology and bioinformatics departments should help facilitate the development of computational pathology.
    Archives of pathology & laboratory medicine 06/2015; DOI:10.5858/arpa.2015-0093-SA · 2.84 Impact Factor
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    ABSTRACT: DNA analysis by NGS has become important to direct the clinical care of cancer patients. However, NGS is not successful in all cases, and the factors responsible for test failures have not been systematically evaluated. A series of 1528 solid and hematolymphoid tumor specimens was tested by an NGS comprehensive cancer panel during 2012-2014. DNA was extracted and 2×101 bp paired-end sequence reads were generated on cancer-related genes utilizing Illumina HiSeq and MiSeq platforms. Testing was unsuccessful in 343 (22.5%) specimens. The failure was due to insufficient tissue (INST) in 223/343 (65%) cases, insufficient DNA (INS-DNA) in 99/343 (28.9%) cases, and failed library (FL) in 21/343 (6.1%) cases. 87/99 (88%) of the INS-DNA cases had below 10 ng DNA available for testing. Factors associated with INST and INS-DNA failures were site of biopsy (SOB) and type of biopsy (TOB) (both p < 0.0001), and clinical setting of biopsy (CSB, initial diagnosis or recurrence) (p < 0.0001). Factors common to INST and FL were age of specimen (p ≤ 0.006) and tumor viability (p ≤ 0.05). Factors common to INS-DNA and FL were DNA purity and DNA degradation (all p ≤ 0.005). In multivariate analysis, common predictors for INST and INS-DNA included CSB (p = 0.048 and p < 0.0001) and TOB (both p ≤ 0.003), respectively. SOB (p = 0.004) and number of cores (p = 0.001) were specific for INS-DNA, whereas TOB and DNA degradation were associated with FL (p = 0.04 and 0.02, respectively). Pre-analytical causes (INST and INS-DNA) accounted for about 90% of all failed cases; independent of test design. Clinical setting; site and type of biopsy; and number of cores used for testing all correlated with failure. Accounting for these factors at the time of tissue biopsy acquisition could improve the analytic success rate. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    Molecular oncology 05/2015; DOI:10.1016/j.molonc.2015.05.004 · 5.33 Impact Factor
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    ABSTRACT: The information-theoretic concept of Shannon entropy can be used to quantify the information provided by a diagnostic test. We hypothesized that in tumor types with stereotyped mutational profiles, the results of NGS testing would yield lower average information than in tumors with more diverse mutations. To test this hypothesis, we estimated the entropy of NGS testing in various cancer types, using results obtained from clinical sequencing. A set of 238 tumors were subjected to clinical targeted NGS across all exons of 27 genes. There were 120 actionable variants in 109 cases, occurring in the genes KRAS, EGFR, PTEN, PIK3CA, KIT, BRAF, NRAS, IDH1, and JAK2. Sequencing results for each tumor were modeled as a dichotomized genotype (actionable mutation detected or not detected) for each of the 27 genes. Based upon the entropy of these genotypes, sequencing was most informative for colorectal cancer (3.235 bits of information/case) followed by high grade glioma (2.938 bits), lung cancer (2.197 bits), pancreatic cancer (1.339 bits), and sarcoma/STTs (1.289 bits). In the most informative cancer types, the information content of NGS was similar to surgical pathology examination (modeled at approximately 2-3 bits). Entropy provides a novel measure of utility for laboratory testing in general and for NGS in particular. This metric is, however, purely analytical and does not capture the relative clinical significance of the identified variants, which may also differ across tumor types. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cancer Genetics 05/2015; 208(9). DOI:10.1016/j.cancergen.2015.05.030 · 2.98 Impact Factor
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    ABSTRACT: The BRAF mutation occurs commonly in papillary thyroid carcinoma (PTC). Previous investigations of its utility to predict recurrence-free survival (RFS) and disease-specific survival (DSS) have reported conflicting results and its role remains unclear. The purpose of this retrospective study was to determine the incidence of the BRAF mutation and analyze its relationship to clinicopathologic risk factors and long-term outcomes in the largest, single-institution American cohort to date. BRAF mutational status was determined in 508 PTC patients using RFLP analysis. The relationships between BRAF mutation status, patient and tumor characteristics, RFS, and DSS were analyzed. The BRAF mutation was present in 67% of patients. On multivariate analysis, presence of the mutation predicted only for capsular invasion (HR, 1.7; 95% CI, 1.1-2.6), cervical lymph node involvement (HR, 1.7; 95% CI, 1.1-2.7), and classic papillary histology (HR, 1.8; 95% CI 1.1-2.9). There was no significant relationship between the BRAF mutation and RFS or DSS, an observation that was consistent across univariate, multivariate, and Kaplan-Meier analyses. This is the most extensive study to date in the United States to demonstrate that BRAF mutation is of no predictive value for recurrence or survival in PTC. We found correlations of BRAF status and several clinicopathologic characteristics of high-risk disease, but limited evidence that the mutation correlates with more extensive or aggressive disease. This analysis suggests that BRAF is minimally prognostic in PTC. However, prevalence of the BRAF mutation is 70% in the general population, providing the opportunity for targeted therapy. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
    Cancer Medicine 02/2015; 4(6). DOI:10.1002/cam4.417 · 2.50 Impact Factor
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    ABSTRACT: Context .- Genomic sequencing for cancer is offered by commercial for-profit laboratories, independent laboratory networks, and laboratories in academic medical centers and integrated health networks. The variability among the tests has created a complex, confusing environment. Objective .- To address the complexity, the Personalized Health Care (PHC) Committee of the College of American Pathologists proposed the development of a cancer genomics resource list (CGRL). The goal of this resource was to assist the laboratory pathology and clinical oncology communities. Design .- The PHC Committee established a working group in 2012 to address this goal. The group consisted of site-specific experts in cancer genetic sequencing. The group identified current next-generation sequencing (NGS)-based cancer tests and compiled them into a usable resource. The genes were annotated by the working group. The annotation process drew on published knowledge, including public databases and the medical literature. Results .- The compiled list includes NGS panels offered by 19 laboratories or vendors, accompanied by annotations. The list has 611 different genes for which NGS-based mutation testing is offered. Surprisingly, of these 611 genes, 0 genes were listed in every panel, 43 genes were listed in 4 panels, and 54 genes were listed in 3 panels. In addition, tests for 393 genes were offered by only 1 or 2 institutions. Table 1 provides an example of gene mutations offered for breast cancer genomic testing with the annotation as it appears in the CGRL 2014. Conclusions .- The final product, referred to as the Cancer Genomics Resource List 2014, is available as supplemental digital content.
    Archives of pathology & laboratory medicine 12/2014; 139(8). DOI:10.5858/arpa.2014-0330-CP · 2.84 Impact Factor
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    ABSTRACT: BACKGROUNDA clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non–small cell lung cancer (NSCLC).METHODS Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists–accredited, Clinical Laboratory Improvement Amendments–certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS.RESULTSTwo hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients.CONCLUSIONSNGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing. Cancer 2014. © 2014 American Cancer Society.
    Cancer 10/2014; 121(4). DOI:10.1002/cncr.29089 · 4.89 Impact Factor
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    Dataset: mmc1
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    Dataset: mmc3
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    Dataset: mmc4
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    Dataset: mmc2
  • International journal of radiation oncology, biology, physics 09/2014; 90(1):S93. DOI:10.1016/j.ijrobp.2014.05.492 · 4.26 Impact Factor
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    ABSTRACT: Purpose: The practice of “genomic” (or “personalized”) medicine requires the availability of appropriate diagnostic testing. Our study objective was to identify the reasons for health systems to bring next-generation sequencing into their clinical laboratories and to understand the process by which such decisions were made. Such information may be of value to other health systems seeking to provide next-generation sequencing testing to their patient populations. Methods: A standardized open-ended interview was conducted with the laboratory medical directors and/or department of pathology chairs of 13 different academic institutions in 10 different states. Results: Genomic testing for cancer dominated the institutional decision making, with three primary reasons: more effective delivery of cancer care, the perceived need for institutional leadership in the field of genomics, and the premise that genomics will eventually be cost-effective. Barriers to implementation included implementation cost; the time and effort needed to maintain this newer testing; challenges in interpreting genetic variants; establishing the bioinformatics infrastructure; and curating data from medical, ethical, and legal standpoints. Ultimate success depended on alignment with institutional strengths and priorities and working closely with institutional clinical programs. Conclusion: These early adopters uniformly viewed genomic analysis as an imperative for developing their expertise in the implementation and practice of genomic medicine.
    Genetics in medicine: official journal of the American College of Medical Genetics 07/2014; 16(12). DOI:10.1038/gim.2014.60 · 7.33 Impact Factor
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    ABSTRACT: Background Clinical outcome of papillary thyroid carcinoma (PTC) in children differs significantly from that of adults. There is no clear explanation of this difference although previous studies have demonstrated a lower prevalence of the BRAFV600E mutation in PTC of children. However, data are limited due to the rarity of this diagnosis. BRAFV600E mutation prevalence and its relationship with outcome in pediatric PTC remain unclear.ProcedureBRAFV600E mutational status was determined in 27 PTC patients less than 22 years of age using restriction fragment length polymorphism (RFLP) analysis. The relationship between BRAFV600E mutation status, patient and tumor characteristics as well as progression-free survival (PFS) were analyzed.ResultsBRAFV600E was present in 63% of patients and occurred more often in male patients versus females (P = 0.033). Presence of the mutation did not correlate with any difference in extent of disease at diagnosis, tumor size, capsular invasion, vascular invasion, soft tissue invasion, or margin status. At 10 years, PFS for BRAFV600E positive versus negative patients was 55.5% versus 70.0%, respectively (P = 0.48). Overall survival was 100% and median follow-up was 13.9 years.Conclusions This study of pediatric PTC demonstrates that BRAFV600E mutations occur in children at a rate comparable to adults. We found a correlation of BRAFV600E with the male gender, but no evidence that the mutation correlates with more extensive or aggressive disease. This analysis suggests that differences in disease course of PTC in children versus adults are not strongly dependent upon the presence of the BRAFV600E mutation. Pediatr Blood Cancer © 2014 Wiley Periodicals, Inc.
    Pediatric Blood & Cancer 07/2014; 61(7). DOI:10.1002/pbc.24935 · 2.39 Impact Factor
  • Alejandro A Gru · Nils Becker · Louis P Dehner · John D Pfeifer
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    ABSTRACT: Although the presence of the t(12;22)(q13;q12) translocation (the defining molecular feature of malignant melanoma of soft parts/clear cell sarcoma) in cutaneous melanoma has been investigated, no large-scale studies have been performed among mucosal melanoma (MucM). In this study we assessed the prevalence of the EWSR1 rearrangement in primary MucM, and analyzed gross and microscopic features with their potential impact on diagnosis and prognosis. Overall, 132 specimens from 84 patients were included. A total of 55 cases had an intramucosal component. Survival of MucMs of the head and neck was associated with two independent factors: size and histology. Tumors more than 3 cm in greatest dimension had an average survival of 12.75 months; those 3 cm or less had an average survival of 38.3 months (P=0.035). Purely epithelioid tumors had an average worse survival of 16.8 months (P=0.028). A cut-off value of 1 mm for Breslow depth provided a statistically significant difference in survival at both 3 and 5 years (P=-0.02) by multivariate analysis in the gynecologic tract. At the molecular level three cases had a EWSR1 rearrangement by fluorescent in-situ hybridization, but only one with an intramucosal component. None of the 58 cases tested by PCR showed the presence of the EWSR1 rearrangement. With the exception of vulvar melanomas, the prognosis of mucosal-associated melanomas was poor and there was a suggestion that spindle morphology may be more favorable. Our study also showed that the EWSR1 rearrangement was very uncommon among MucM. Though 'clear cell sarcoma' is embedded in the sarcoma literature, the synonym 'melanoma of soft parts' has considerable justification in light of our evolving understanding of the molecular genetics in the family of malignant melanomas.
    Melanoma Research 05/2014; 24(4). DOI:10.1097/CMR.0000000000000082 · 2.28 Impact Factor
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    ABSTRACT: The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.
    The Journal of molecular diagnostics: JMD 05/2014; 16(4). DOI:10.1016/j.jmoldx.2014.03.006 · 4.85 Impact Factor
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    ABSTRACT: Context: Genomic medicine is revolutionizing patient care. Physicians in areas as diverse as oncology, obstetrics, and infectious disease have begun using next-generation sequencing assays as standard diagnostic tools. Objective: To review the role of pathologists in genomic testing as well as current educational programs and future training needs in genomic pathology. Data sources: Published literature as well as personal experience based on committee membership and genomic pathology curricular design. Conclusions: Pathologists, as the directors of the clinical laboratories, must be prepared to integrate genomic testing into their practice. The pathology community has made significant progress in genomics-related education. A continued coordinated and proactive effort will ensure a future vital role for pathologists in the evolving health care system and also the best possible patient care.
    Archives of pathology & laboratory medicine 04/2014; 138(4):498-504. DOI:10.5858/arpa.2013-0359-SA · 2.84 Impact Factor
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    ABSTRACT: Precision medicine uses individually determined genomic information to guide treatment in cancer and other diseases. We have implemented a clinical genomics assay that uses targeted next-generation sequencing of 25 cancer-related genes to guide the use of targeted therapies in diverse malignancies. We report the case of a 55-year-old woman with a poorly differentiated squamous cell carcinoma of thymic origin, with disease progression after standard treatment. Targeted tumor sequencing revealed the presence of a KIT codon 579 deletion (p.D579del). This specific mutation has not previously been associated with thymic tumors, but has been reported in gastrointestinal stromal tumors and has been associated with response to imatinib. Imatinib therapy was instituted for and resulted in stabilization of disease. This case illustrates the potential of clinical next-generation sequencing to open unexpected avenues for treatment and thereby improve patient outcomes.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 02/2014; 9(2):e12-6. DOI:10.1097/JTO.0b013e3182a7d22e · 5.28 Impact Factor
  • Johann D Hertel · Phyllis C Huettner · John D Pfeifer
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    ABSTRACT: The microcystic elongated and fragmented (MELF) pattern of myoinvasion is a feature of some well-differentiated endometrial endometrioid adenocarcinomas that has been associated with poor prognosis. The myoinvasion in MELF-pattern tumors can be subtle and lead to underestimation of the depth of myometrial invasion resulting in tumor understaging; the presence of lymphvascular space invasion (LVSI) and lymph node metastasis in MELF-pattern tumors can also be subtle and lead to tumor understaging. To investigate the association of MELF-pattern invasion and lymph node metastasis, we reviewed a series of well-differentiated endometrioid adenocarcinomas and correlated the presence of MELF-pattern myoinvasion and LVSI with lymph node metastasis. Cases of T1 stage well-differentiated endometrioid adenocarcinomas with LVSI and a concurrent lymph node dissection were identified from departmental files. Hematoxylin and eosin-stained slides from the hysterectomy specimen and lymph nodes were reviewed for the presence of MELF-pattern myoinvasion, LVSI, and nodal metastasis. MELF-pattern myoinvasion was identified at least focally in 36% of cases. The pattern of LVSI differed between cases with MELF-pattern invasion and conventional-type invasion, as did the pattern of nodal metastasis. A statistically significantly higher rate of lymph node metastasis was present in cases with MELF-pattern invasion than in cases with conventional invasion, and the rate stratified with the proportion of MELF-pattern adenocarcinomas. MELF-pattern cases carry an increased rate of lymph node metastasis even within the subset of endometrioid tumors with LVSI, which has implications in routine clinical practice as it signals the importance of recognizing MELF-pattern myoinvasion.
    International journal of gynecological pathology: official journal of the International Society of Gynecological Pathologists 01/2014; 33(2). DOI:10.1097/PGP.0b013e318285657b · 1.67 Impact Factor
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    ABSTRACT: Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture-enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic and/or staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.
    The American journal of surgical pathology 01/2014; 38(4). DOI:10.1097/PAS.0000000000000161 · 5.15 Impact Factor

Publication Stats

3k Citations
514.92 Total Impact Points


  • 2015
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1992–2015
    • Washington University in St. Louis
      • • Department of Pathology and Immunology
      • • Department of Obstetrics and Gynecology
      • • Department of Molecular Microbiology
      San Luis, Missouri, United States
  • 2002–2007
    • Barnes Jewish Hospital
      San Luis, Missouri, United States
  • 2005
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada
  • 2001
    • Johns Hopkins Medicine
      Baltimore, Maryland, United States
  • 2000
    • St. Jude Children's Research Hospital
      • Department of Pathology
      Memphis, Tennessee, United States
  • 1993–1997
    • Lund University
      Lund, Skåne, Sweden
  • 1995
    • Case Western Reserve University
      • Institute of Pathology
      Cleveland, Ohio, United States