John D Pfeifer

Harvard Medical School, Boston, Massachusetts, United States

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Publications (143)517.76 Total impact

  • Shashikant Kulkarni · John Pfeifer ·
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    ABSTRACT: The DNA sequencing platforms that are currently in widespread use to perform massively parallel sequencing, which as a group are currently referred to as next-generation sequencing (NGS) platforms, have enabled the genomic revolution in science and medicine. However, current NGS platforms do not represent the final stage of development of DNA sequencing technologies. A number of so-called third-generation approaches, which are already available commercially, make it possible to sequence individual DNA molecules without the need for library amplification steps. These approaches offer a number of advantages over current NGS methods including avoidance of the artifactual DNA mutations and strand biases introduced by even limited cycles of PCR; higher throughput and faster turnaround times, longer read lengths (by some platforms) that enhance de novo contig and genome assembly; higher consensus accuracy; and analysis of smaller quantities of nucleic acids which is a clear advantage in clinical settings. However, the third-generation approaches are themselves transitional to fourth-generation techniques that, while largely still in developmental phases, rely on entirely different principles of chemistry and physics to produce DNA sequence. While these fourth-generation technologies are years away from widespread clinical use, they provide a glimpse into the ever more sophisticated utilization of synthetic materials and advanced electronics that will continue to make DNA sequence analysis even faster and less costly.
    Clinical Genomics, 12/2015: pages 69-76; , ISBN: 9780124047488
  • Haley Abel · John Pfeifer · Eric Duncavage ·
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    ABSTRACT: Identification of recurrent chromosomal rearrangements or translocations is critically important for the diagnosis and prognosis of both hematologic malignancies and solid tumors. In the clinical laboratory setting, detection of rearrangements is typically performed by routine cytogenetics or interphase/metaphase fluorescence in situ hybridization (FISH). However, next-generation sequencing (NGS)-based methods are also capable of detecting chromosomal rearrangements in both the clinical and research settings. This chapter will introduce NGS methods to detect recurrent rearrangements from whole genome sequencing data, targeted sequencing data, and RNA sequencing data. It will review various basic approaches including discordant paired-end and single-end (split-end) read mapping and associated informatic tools for translocation detection. Further emphasis will be placed on the clinical performance of such tools including the sensitivity and specificity of rearrangement detection from targeted DNA sequencing data.
    Clinical Genomics, 12/2015: pages 151-164; , ISBN: 9780124047488
  • David H. Spencer · Bin Zhang · John Pfeifer ·
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    ABSTRACT: Single nucleotide variants (SNVs) occur when a single nucleotide (e.g., A, T, C, or G) is altered in the DNA sequence. SNVs are by far the most common type of sequence change, and there are a number of endogenous and exogenous sources of damage that lead to the single base pair substitution mutations that create SNVs. The biologic impact of SNVs in coding regions depends on their type (synonymous versus missense), and in noncoding regions depends on their impact on RNA processing or gene regulation. Nonetheless, selection pressure reduces the overall frequency of single base pair substitutions in coding DNA and in associated regulatory sequences, with the result that the overall SNV rate in coding DNA is much less than that of noncoding DNA. The utility of a clinical next generation sequencing (NGS) assay designed to detect SNVs depends on assay design features including an amplification-based versus hybrid capture-based targeted approach, DNA library complexity, depth of sequencing, tumor cellularity (in sequencing of cancer specimens), specimen fixation, and sequencing platform. From a bioinformatic perspective, many popular NGS analysis programs for SNV detection are designed for constitutional genome analysis where variants occur in either 50% (heterozygous) or 100% (homozygous) of the reads; these prior probabilities are often built-in to the algorithms, and consequently SNVs with variant allele frequencies (VAFs) falling too far outside the expected range for homozygous and heterozygous variants are often ignored as false positives. Thus, sensitive and specific bioinformatic approaches for acquired SNVs require either significant revision of the software packages designed for constitutional testing or new algorithms altogether. Some bioinformatic tools are optimized for very sensitive detection of SNVs in NGS data, but these tools require high coverage depth for acceptable performance and rely on spike-in control samples in order to calibrate run-dependent error models, features that must be accounted for in assay design. There are a number of online tools that can be used to predict the impact of an SNV and evaluate whether an SNV has a documented disease association. Guidelines for reporting SNVs detected in constitutional NGS testing have been developed; consensus guidelines for reporting somatic or acquired SNVs are under development.
    Clinical Genomics, 12/2015: pages 109-127; , ISBN: 9780124047488

  • Archives of pathology & laboratory medicine 11/2015; 139(11):1400-1412. DOI:10.5858/arpa.2014-0568-CP · 2.84 Impact Factor
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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
  • Hussam Al-Kateb · TuDung T Nguyen · Karen Steger-May · John D Pfeifer ·
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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
  • Ian S. Hagemann · Patrick K. O’Neill · Ivan Erill · John D. Pfeifer ·
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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
  • Shashikant Kulkarni · John Pfeifer ·
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    ABSTRACT: Next generation sequencing (NGS) is subject to the same regulatory standards as other molecular genetic tests. The regulatory oversight of clinical laboratories and of clinical NGS testing varies worldwide, but many countries base compliance on standards set by the International Standards Organization (ISO). In the USA, oversight is federally regulated based on the Clinical Laboratory Improvement Amendments act of 1988 (CLIA '88). In addition, in the USA, the Food and Drug Administration (FDA) regulates the manufacture of equipment, devices, and assay reagent kits used in clinical testing, which in the context of NGS includes the sequencing platforms themselves, the kits used for library preparation and specific tests, and the bioinformatics pipelines to analyze the data; most NGS tests are categorized as laboratory developed tests (LDTs), which are also subject to FDA oversight. Regardless of the regulatory standards, as with other laboratory tests, NGS has a test cycle that includes a preanalytic phase, an analytic phase, and a postanalytic phase, and the quality control (QC) and quality assurance (QA) principles that govern these phases for more traditional molecular genetic tests are also applicable to NGS tests. However, NGS tests are somewhat unique in that the analytic portion of the test consists of three individual components, specifically the sequence platform itself; the so-called wet-bench procedures that are involved in DNA library preparation; and the bioinformatics associated with base calling, reference genome alignment, variant identification, variant annotation, and variant interpretation. The fact that there are three independent aspects of NGS complicates proficiency testing (PT) of NGS assays. While the emphasis to date has been on the development of comprehensive PT surveys that evaluate all three aspects of an NGS test, a novel type of PT has recently been developed (termed in silico-based PT) in order to specifically address the bioinformatics component of the tests.
    Clinical Genomics, 01/2015: pages 377-391; , ISBN: 9780124047488
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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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  • 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

Publication Stats

4k Citations
517.76 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
  • 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