Bing Melody Zhang

All Publications

• Minimal/measurable residual disease (MRD) monitoring in patients with lymphoid neoplasms by high-throughput sequencing of the T-cell receptor. The Journal of molecular diagnostics : JMD Tung, J. K., Jangam, D., Ho, C. C., Fung, E., Khodadoust, M. S., Kim, Y. H., Zehnder, J. L., Stehr, H., Zhang, B. M. 2023

  Abstract

  High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies seen at our institution. A custom bioinformatics pipeline and database was also developed to facilitate MRD analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 out of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.

  View details for DOI 10.1016/j.jmoldx.2023.02.002

  View details for PubMedID 36870603

• The role of CD8+ T cell clones in immune thrombocytopenia. Blood Malik, A., Sayed, A. A., Han, P., Tan, M. M., Watt, E., Constantinescu-Bercu, A., Cocker, A. T., Khoder, A., Saputil, R. C., Thorley, E. V., Teklemichael, A., Ding, Y., Hart, A. C., Zhang, H., Mitchell, W. A., Imami, N., Crawley, J. T., Salles-Crawley, I. I., Bussel, J. B., Zehnder, J. L., Adams, S. P., Zhang, B. M., Cooper, N. 2023

  Abstract

  Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

  View details for DOI 10.1182/blood.2022018380

  View details for PubMedID 36749920

• Identification of a pathogenic TUBB1 variant in a Chinese family with congenital macrothrombocytopenia through whole genome sequencing. Platelets Hou, Y. n., Shao, L. n., Zhou, H. n., Liu, Y. n., Fisk, D. G., Spiteri, E. n., Zehnder, J. L., Peng, J. n., Zhang, B. M., Hou, M. n. 2021: 1–5

  Abstract

  Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We herein report a large Chinese family presented with phenotypic variability involving thrombocytopenia and/or giant platelets. Whole genome sequencing (WGS) of the proband and one of his affected brothers identified a potentially pathogenic c.952 C > T heterozygous variant in the TUBB1 gene. This p.R318W β1-tubulin variant was also identified in three additional siblings and five members of the next generation. These findings were consistent with an autosomal dominant inheritance with incomplete penetrance. Moreover, impaired platelet agglutination in response to ristocetin was detected in the patient's brother. Half of the family members harboring the p.R318W mutation displayed significantly decreased external release of p-selectin by stimulated platelets. The p.R318W β1-tubulin mutation was identified for the first time in a Chinese family with congenital macrothrombocytopenia using WGS as an unbiased sequencing approach. Affected individuals within the family demonstrated impaired platelet aggregation and/or release functions.

  View details for DOI 10.1080/09537104.2020.1869714

  View details for PubMedID 33400601

• Validation of a Next-Generation Sequencing-based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing. The Journal of molecular diagnostics : JMD Ho, C. C., Tung, J. K., Zehnder, J. L., Zhang, B. M. 2021

  Abstract

  Assessment of T-cell receptor gamma (TRG) gene rearrangement is an important consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard for such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment-size based methods since they generate results with specific clonotype sequence information and allow for more accurate quantitation. We therefore evaluated various test parameters and performance characteristics for a commercially available NGS-based TRG gene rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%, as compared to the CE-based assay. The concordance rate was 88∼95% for Vγ1-8, Vγ10 and Vγ11 gene families, but lower for the Vγ9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing different clonotypes with the same fragment size. Our clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay.

  View details for DOI 10.1016/j.jmoldx.2021.03.008

  View details for PubMedID 33892183

• Specifications of the variant curation guidelines for ITGA2B/ITGB3: ClinGen Platelet Disorder Variant Curation Panel. Blood advances Ross, J. E., Zhang, B. M., Lee, K. n., Mohan, S. n., Branchford, B. R., Bray, P. n., Dugan, S. N., Freson, K. n., Heller, P. G., Kahr, W. H., Lambert, M. P., Luchtman-Jones, L. n., Luo, M. n., Perez Botero, J. n., Rondina, M. T., Ryan, G. n., Westbury, S. n., Bergmeier, W. n., Di Paola, J. n. 2021; 5 (2): 414–31

  Abstract

  Accurate and consistent sequence variant interpretation is critical to the correct diagnosis and appropriate clinical management and counseling of patients with inherited genetic disorders. To minimize discrepancies in variant curation and classification among different clinical laboratories, the American College of Medical Genetics and Genomics (ACMG), along with the Association for Molecular Pathology (AMP), published standards and guidelines for the interpretation of sequence variants in 2015. Because the rules are not universally applicable to different genes or disorders, the Clinical Genome Resource (ClinGen) Platelet Disorder Expert Panel (PD-EP) has been tasked to make ACMG/AMP rule specifications for inherited platelet disorders. ITGA2B and ITGB3, the genes underlying autosomal recessive Glanzmann thrombasthenia (GT), were selected as the pilot genes for specification. Eight types of evidence covering clinical phenotype, functional data, and computational/population data were evaluated in the context of GT by the ClinGen PD-EP. The preliminary specifications were validated with 70 pilot ITGA2B/ITGB3 variants and further refined. In the final adapted criteria, gene- or disease-based specifications were made to 16 rules, including 7 with adjustable strength; no modification was made to 5 rules; and 7 rules were deemed not applicable to GT. Employing the GT-specific ACMG/AMP criteria to the pilot variants resulted in a reduction of variants classified with unknown significance from 29% to 20%. The overall concordance with the initial expert assertions was 71%. These adapted criteria will serve as guidelines for GT-related variant interpretation to increase specificity and consistency across laboratories and allow for better clinical integration of genetic knowledge into patient care.

  View details for DOI 10.1182/bloodadvances.2020003712

  View details for PubMedID 33496739

• An Overview of Characteristics of Clinical Next-Generation Sequencing-Based Testing for Hematologic Malignancies. Archives of pathology & laboratory medicine Zhang, B. M., Keegan, A. n., Li, P. n., Lindeman, N. I., Nagarajan, R. n., Routbort, M. J., Vasalos, P. n., Kim, A. S., Merker, J. D. 2021

  Abstract

  With the increasing integration of molecular alterations into the evaluation of hematologic malignancies (HM), somatic mutation profiling by next-generation sequencing (NGS) has become a common clinical testing strategy. Limited data are available about the characteristics of these assays.To describe assay characteristics, specimen requirements, and reporting practices for NGS-based HM testing using College of American Pathologists proficiency testing survey data.The College of American Pathologists NGS Hematologic Malignancies Survey (NGSHM) results from 78 laboratories were used to determine laboratory practices in NGS-based HM testing.The majority of laboratories performed tumor-only (88.5% [69 of 78]), targeted sequencing of cancer genes or mutation hotspots (98.7% [77 of 78]); greater than 90% performed testing on fresh bone marrow and peripheral blood. The majority of laboratories reported a 5% lower limit of detection for single-nucleotide variants (73.1% [57 of 78]) and small insertions and deletions (50.6% [39 of 77]). A majority of laboratories used benchtop sequencers and custom enrichment approaches.This manuscript summarizes the characteristics of clinical NGS-based testing for the detection of somatic variants in HM. These data may be broadly useful to inform laboratory practice and quality management systems, regulation, and oversight of NGS testing, and precision medicine efforts using a data-driven approach.

  View details for DOI 10.5858/arpa.2019-0661-CP

  View details for PubMedID 33450747

• HLA-haplotype loss after TCRalphabeta/CD19-depleted haploidentical HSCT. Bone marrow transplantation Shyr, D. C., Zhang, B. M., Saini, G., Madani, N. D., Schultz, L. M., Patel, S., Kristovich, K., Fernandez-Vina, M., Bertaina, A. 2020

  View details for DOI 10.1038/s41409-020-01081-0

  View details for PubMedID 33070150

• IDH2 Mutation in a Patient with Metastatic Colon Cancer NEW ENGLAND JOURNAL OF MEDICINE Zhang, B. M., Zehnder, J. L., Suarez, C. J. 2017; 376 (20): 1991-1992

  View details for DOI 10.1056/NEJMc1701072

  View details for PubMedID 28514606

• A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia. Blood Zhang, B., Ng, D., Jones, C., Oh, S. T., Nolan, G. P., Salehi, S., Wong, W., Zehnder, J. L., Gotlib, J. 2011; 118 (26): 6988-6990

  View details for DOI 10.1182/blood-2011-10-386177

  View details for PubMedID 22194398

• The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP BLOOD Zhang, B., Lo, C., Shen, L., Sood, R., Jones, C., Cusmano-Ozog, K., Park-Snyder, S., Wong, W., Jeng, M., Cowan, T., Engleman, E. G., Zehnder, J. L. 2011; 117 (17): 4569-4579

  Abstract

  Pediatric immune thrombocytopenia (ITP) is usually self-limited. However, approximately 20% of children develop chronic ITP, which can be associated with significant morbidity because of long-term immunosuppression and splenectomy in refractory cases. To explore the molecular mechanism of chronic ITP compared with acute ITP, we studied 63 pediatric patients with ITP. Gene expression analysis of whole blood revealed distinct signatures for acute and chronic ITP. Oxidative stress-related pathways were among the most significant chronic ITP-associated pathways. Overexpression of VNN1, an oxidative stress sensor in epithelial cells, was most strongly associated with progression to chronic ITP. Studies of normal persons demonstrated VNN1 expression in a variety of blood cells. Exposure of blood mononuclear cells to oxidative stress inducers elicited dramatic up-regulation of VNN1 and down-regulation of PPARγ, indicating a role for VNN1 as a peripheral blood oxidative stress sensor. Assessment of redox state by tandem mass spectrometry demonstrated statistically significant lower glutathione ratios in patients with ITP versus healthy controls; lower glutathione ratios were also seen in untreated patients with ITP compared with recently treated patients. Our work demonstrates distinct patterns of gene expression in acute and chronic ITP and implicates oxidative stress pathways in the pathogenesis of chronic pediatric ITP.

  View details for DOI 10.1182/blood-2010-09-304931

  View details for PubMedID 21325602

• A human autoimmune organoid model reveals IL-7 function in coeliac disease. Nature Santos, A. J., van Unen, V., Lin, Z., Chirieleison, S. M., Ha, N., Batish, A., Chan, J. E., Cedano, J., Zhang, E. T., Mu, Q., Guh-Siesel, A., Tomaske, M., Colburg, D., Varma, S., Choi, S. S., Christophersen, A., Baghdasaryan, A., Yost, K. E., Karlsson, K., Ha, A., Li, J., Dai, H., Sellers, Z. M., Chang, H. Y., Dunn, J. C., Zhang, B. M., Mellins, E. D., Sollid, L. M., Fernandez-Becker, N. Q., Davis, M. M., Kuo, C. J. 2024

  Abstract

  In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

  View details for DOI 10.1038/s41586-024-07716-2

  View details for PubMedID 39048815

• Exploring Pattern of Relapse in Pediatric Patients with Acute Lymphocytic Leukemia and Acute Myeloid Leukemia Undergoing Stem Cell Transplant Using Machine Learning Methods. Journal of clinical medicine Shyr, D., Zhang, B. M., Saini, G., Brewer, S. C. 2024; 13 (14)

  Abstract

  Background. Leukemic relapse remains the primary cause of treatment failure and death after allogeneic hematopoietic stem cell transplant. Changes in post-transplant donor chimerism have been identified as a predictor of relapse. A better predictive model of relapse incorporating donor chimerism has the potential to improve leukemia-free survival by allowing earlier initiation of post-transplant treatment on individual patients. We explored the use of machine learning, a suite of analytical methods focusing on pattern recognition, to improve post-transplant relapse prediction. Methods. Using a cohort of 63 pediatric patients with acute lymphocytic leukemia (ALL) and 46 patients with acute myeloid leukemia (AML) who underwent stem cell transplant at a single institution, we built predictive models of leukemic relapse with both pre-transplant and post-transplant patient variables (specifically lineage-specific chimerism) using the random forest classifier. Local Interpretable Model-Agnostic Explanations, an interpretable machine learning tool was used to confirm our random forest classification result. Results. Our analysis showed that a random forest model using these hyperparameter values achieved 85% accuracy, 85% sensitivity, 89% specificity for ALL, while for AML 81% accuracy, 75% sensitivity, and 100% specificity at predicting relapses within 24 months post-HSCT in cross validation. The Local Interpretable Model-Agnostic Explanations tool was able to confirm many variables that the random forest classifier identified as important for the relapse prediction. Conclusions. Machine learning methods can reveal the interaction of different risk factors of post-transplant leukemic relapse and robust predictions can be obtained even with a modest clinical dataset. The random forest classifier distinguished different important predictive factors between ALL and AML in our relapse models, consistent with previous knowledge, lending increased confidence to adopting machine learning prediction to clinical management.

  View details for DOI 10.3390/jcm13144021

  View details for PubMedID 39064061

  View details for PubMedCentralID PMC11277799

• Implementation, Evolution, and Laboratory Performance of Methods-Based Proficiency Testing for Next-Generation Sequencing Detection of Germline Sequence Variants. Archives of pathology & laboratory medicine Tsuchiya, K. D., Funke, B., Hegde, M., Santani, A., Souers, R. J., Szelinger, S., Halley, J., Zhao, Q., Mot, N., Roy, A., Smith, V. L., Zhang, B. M., Voelkerding, K., Moyer, A. M. 2023

  Abstract

  Next-generation sequencing (NGS)-based assays are used for diagnosis of diverse inherited disorders. Limited data are available pertaining to interlaboratory analytical performance of these assays.To report on the College of American Pathologists (CAP) NGS Germline Program, which is methods based, and explore the evolution in laboratory testing practices.Results from the NGS Germline Program from 2016-2020 were analyzed for interlaboratory analytical performance. Self-reported laboratory testing practices were also evaluated.From 2016-2020, a total of 297 laboratories participated in at least 1 program mailing. Of the 289 laboratories that provided information on tests offered, 138 (47.8%) offered only panel testing throughout their enrollment, while 35 (12.1%) offered panels and exome testing, 30 (10.4%) offered only exomes, 9 (3.1%) offered only genomes, and 15 (5.2%) offered panels, exomes, and genomes. The remainder (62 laboratories, 21.4%) changed their test offerings during the 2016-2020 timeframe. Considering each genomic position/interval, the median detection percentage at variant positions across the 2016-2020 mailings ranged from 94.3% to 100%, while at reference positions (no variant detected), the median correct response percentage was 100% across all mailings. When considering performance of individual laboratories, 89.5% (136 of 152) to 98.0% (149 of 152) of laboratories successfully met the detection threshold (≥90% of the variants present), while 94.6% (87 of 92) to 100% (163 of 163) of laboratories met the 95% specificity threshold across mailings.Since the inception of this program, laboratories have consistently performed well. The median sensitivity and specificity of detection of sequence variants included in this program (eg, single nucleotide variants, insertions, and deletions) were 100.0%.

  View details for DOI 10.5858/arpa.2023-0090-CP

  View details for PubMedID 37852169

• The Association of Class I and II Human Leukocyte Antigen Serotypes With End-Stage Kidney Disease Due to Membranoproliferative Glomerulonephritis and Dense Deposit Disease. American journal of kidney diseases : the official journal of the National Kidney Foundation Afolabi, H., Zhang, B. M., O'Shaughnessy, M., Chertow, G. M., Lafayette, R., Charu, V. 2023

  Abstract

  RATIONALE & OBJECTIVE: Membranoproliferative glomerulonephritis (MPGN), encompassing several distinct diseases, is a rare but significant cause of kidney failure in the US. Potential etiologies of MPGN are unclear, but prior studies have suggested dysregulation of the alternative complement pathway, and recently, autoimmunity as potential mechanisms driving MPGN pathogenesis. In this study, we examined HLA associations with end-stage kidney disease (ESKD) due to MPGN and Dense Deposit Disease (DDD) in a large racially and ethnically diverse US-based cohort.STUDY DESIGN: Case-control study.SETTING &PARTICIPANTS: Using USRDS and UNOS data, we identified 3424 patients with kidney failure due to MPGN and 263 due to DDD. We matched patients to kidney donor controls on designated race and ethnicity in a 1:15 ratio.EXPOSURES: 58 class I and II HLA serotypes.OUTCOMES: Case-control status.ANALYTICAL APPROACH: For each disease cohort, univariable and multivariable logistic regression analyses were used to investigate associations between the disease and 58 HLA serotypes. In subgroup analyses, we investigated HLA associations in White and Black patients. We also studied anti-GBM nephritis as a positive-control outcome. We applied a Bonferroni correction to account for multiple comparisons.RESULTS: Eighteen serotypes were significantly associated with the odds of having MPGN in univariable analyses, with DR17 having the strongest association ([OR]: 1.55, 95% CI: 1.44-1.68; p-value 4.33e-28). No significant associations were found between any HLA serotype and DDD. Designated race-specific analyses showed comparable findings. We recapitulated known HLA associations in anti-GBM nephritis.LIMITATIONS: Reliance on HLA serotypes (rather than genotype), lack of biopsy-confirmed diagnoses.CONCLUSIONS: HLA-DR17 is associated with ESKD due to MPGN in a racially and ethnically diverse cohort. The strength of association was similar in White and Black patients, suggesting a role in the pathogenesis of MPGN. No HLA associations were observed in patients with DDD.

  View details for DOI 10.1053/j.ajkd.2023.06.005

  View details for PubMedID 37739026

• A Primer on Gene Editing: What Does It Mean for Pathologists? Archives of pathology & laboratory medicine Cushman-Vokoun, A., Schmidt, R. J., Hiemenz, M. C., Fung, M., Zhang, B. M., Bradshaw, G., Gandhi, M., Yao, J., Yohe, S., Beckman, A., Grody, W. W., Giannikopoulos, P. 2023

  Abstract

  Gene editing-based therapies are currently in development in the areas of oncology, inherited disease, and infectious disease. These potentially life-altering therapies are derived from decades of research in both academic and industry settings that developed technologies rooted in principles and products of nature. However, with such technologic developments come many important considerations, including adverse risks, high cost, and ethical questions.To educate pathologists about gene editing technologies, inform them of potential indications and risks, outline regulatory and practical issues that could affect hospital-based practice and laboratory testing, and advocate that pathologists need to be present at discussions among industry and regulators pertaining to gene editing-based therapies.A Gene Editing Workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop an educational paper to serve as a stimulus to increase pathologist involvement and inquiry in gene editing therapeutic and diagnostic implementation.Through multiple discussions and literature review, the workgroup identified potential gaps in pathologists' knowledge of gene editing. Additional topics that could impact pathology and laboratory medicine were also identified and summarized in order to facilitate pathologists as stakeholders in gene editing therapy administration and monitoring and potential use in diagnostics.Gene editing therapy is a complex but potentially transformative area of medicine. This article serves as an introduction to pathologists to assist them in future discussions with colleagues and potentially identify and alter pathology practices that relate to gene editing.

  View details for DOI 10.5858/arpa.2022-0410-CP

  View details for PubMedID 37610100

• A Primer on Gene Editing. Archives of pathology & laboratory medicine Cushman-Vokoun, A., Schmidt, R. J., Hiemenz, M. C., Fung, M., Zhang, B. M., Bradshaw, G., Gandhi, M., Yao, J., Yohe, S., Beckman, A., Grody, W. W., Giannikopoulos, P. 2023

  Abstract

  Gene editing-based therapies are currently in development in the areas of oncology, inherited disease, and infectious disease. These potentially life-altering therapies are derived from decades of research in both academic and industry settings that developed technologies rooted in principles and products of nature. However, with such technologic developments come many important considerations, including adverse risks, high cost, and ethical questions.To educate pathologists about gene editing technologies, inform them of potential indications and risks, outline regulatory and practical issues that could affect hospital-based practice and laboratory testing, and advocate that pathologists need to be present at discussions among industry and regulators pertaining to gene editing-based therapies.A Gene Editing Workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop an educational paper to serve as a stimulus to increase pathologist involvement and inquiry in gene editing therapeutic and diagnostic implementation.Through multiple discussions and literature review, the workgroup identified potential gaps in pathologists' knowledge of gene editing. Additional topics that could impact pathology and laboratory medicine were also identified and summarized in order to facilitate pathologists as stakeholders in gene editing therapy administration and monitoring and potential use in diagnostics.Gene editing therapy is a complex but potentially transformative area of medicine. This article serves as an introduction to pathologists to assist them in future discussions with colleagues and potentially identify and alter pathology practices that relate to gene editing.

  View details for DOI 10.5858/arpa.2022-0410-CP

  View details for PubMedID 37603682

• Laboratory perspectives in the development of polygenic risk scores for disease: A points to consider statement of the American College of Medical Genetics and Genomics (ACMG). Genetics in medicine : official journal of the American College of Medical Genetics Reddi, H. V., Wand, H., Funke, B., Zimmermann, M. T., Lebo, M. S., Qian, E., Shirts, B. H., Zou, Y. S., Zhang, B. M., Rose, N. C., Abu-El-Haija, A. 2023: 100804

  View details for DOI 10.1016/j.gim.2023.100804

  View details for PubMedID 36971772

• Class I and II Human Leukocyte Antigen Serotypes Associated with End-Stage Kidney Disease due to Membranoproliferative Glomerulonephritis and Dense Deposit Disease Afolabi, H., Zhang, B., O'Shaughnessy, M., Chertow, G., Lafayette, R., Charu, V. ELSEVIER SCIENCE INC. 2023: S1422-S1423

  View details for Web of Science ID 000990969803123

• HLA-DR/DQ Single Molecule Eplet Mismatch And Formation Of De Novo Donor-Specific Anti-HLA Antibodies After Kidney Transplantation In Children Sigurjonsdottir, V., Piburn, K., Grimm, P., Zhang, B. M. WILEY. 2023

  View details for Web of Science ID 001002465500076

• Primary Graft Dysfunction Is Associated With Development of Early Cardiac Allograft Vasculopathy, but Not Other Immune-mediated Complications, After Heart Transplantation. Transplantation Han, J., Moayedi, Y., Henricksen, E. J., Waddell, K., Valverde-Twiggs, J., Kim, D., Luikart, H., Zhang, B. M., Teuteberg, J., Khush, K. K. 2023

  Abstract

  We investigated associations between primary graft dysfunction (PGD) and development of acute cellular rejection (ACR), de novo donor-specific antibodies (DSAs), and cardiac allograft vasculopathy (CAV) after heart transplantation (HT).A total of 381 consecutive adult HT patients from January 2015 to July 2020 at a single center were retrospectively analyzed. The primary outcome was incidence of treated ACR (International Society for Heart and Lung Transplantation grade 2R or 3R) and de novo DSA (mean fluorescence intensity >500) within 1 y post-HT. Secondary outcomes included median gene expression profiling score and donor-derived cell-free DNA level within 1 y and incidence of cardiac allograft vasculopathy (CAV) within 3 y post-HT.When adjusted for death as a competing risk, the estimated cumulative incidence of ACR (PGD 0.13 versus no PGD 0.21; P = 0.28), median gene expression profiling score (30 [interquartile range, 25-32] versus 30 [interquartile range, 25-33]; P = 0.34), and median donor-derived cell-free DNA levels was similar in patients with and without PGD. After adjusting for death as a competing risk, estimated cumulative incidence of de novo DSA within 1 y post-HT in patients with PGD was similar to those without PGD (0.29 versus 0.26; P = 0.10) with a similar DSA profile based on HLA loci. There was increased incidence of CAV in patients with PGD compared with patients without PGD (52.6% versus 24.8%; P = 0.01) within the first 3 y post-HT.During the first year after HT, patients with PGD had a similar incidence of ACR and development of de novo DSA, but a higher incidence of CAV when compared with patients without PGD.

  View details for DOI 10.1097/TP.0000000000004551

  View details for PubMedID 36801852

• Molecular Diagnosis in Hematology Wintrobe’s Clinical Hematology Zhang, B. M., Zehnder, J. L. 2023; 15th
• CD8(+) TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia Malik, A., Tan, M. H., Sayed, A. A., Han, P., Watt, E., Constantinescu-Bercu, A., Khoder, A., Cocker, A. H., Thorley, E., Teklemichael, A., Ding, Y., Saputil, R. C., Hart, A. J., Zhang, H., Mitchell, W. A., Imami, N., Crawley, J. B., Salles-Crawley, I., Bussel, J. B., Zehnder, J. L., Adams, S., Zhang, B., Cooper, N. AMER SOC HEMATOLOGY. 2022: 2209-2210

  View details for DOI 10.1182/blood-2022-166348

  View details for Web of Science ID 000893223202090

• Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3, and MPL mutations. Blood advances Zhang, H., Chien, M., Hou, Y., Shomali, W., Brar, R., Ho, C., Han, P., Xu, D., Zhang, B. M., Guo, X., Tolentino, L., Wu, N. C., Tsai, A. G., Jin, J., Witteles, W. H., Chen, Z., Abidi, P., Jangam, D., Krieger, M. S., Craig, M., Bussel, J. B., Gotlib, J. R., Zehnder, J. L. 2022

  Abstract

  Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to two patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

  View details for DOI 10.1182/bloodadvances.2021006701

  View details for PubMedID 35381066

• Complement-Binding Donor-Specific Anti-HLA Antibodies: Biomarker for Immunologic Risk Stratification in Pediatric Kidney Transplantation Recipients. Transplant international : official journal of the European Society for Organ Transplantation Sigurjonsdottir, V. K., Purington, N., Chaudhuri, A., Zhang, B. M., Fernandez-Vina, M., Palsson, R., Kambham, N., Charu, V., Piburn, K., Maestretti, L., Shah, A., Gallo, A., Concepcion, W., Grimm, P. C. 2022; 35: 10158

  Abstract

  Antibody-mediated rejection is a common cause of early kidney allograft loss but the specifics of antibody measurement, therapies and endpoints have not been universally defined. In this retrospective study, we assessed the performance of risk stratification using systematic donor-specific antibody (DSA) monitoring. Included in the study were children who underwent kidney transplantation between January 1, 2010 and March 1, 2018 at Stanford, with at least 12-months follow-up. A total of 233 patients were included with a mean follow-up time of 45 (range, 9-108) months. Median age at transplant was 12.3 years, 46.8% were female, and 76% had a deceased donor transplant. Fifty-two (22%) formed C1q-binding de novo donor-specific antibodies (C1q-dnDSA). After a standardized augmented immunosuppressive protocol was implemented, C1q-dnDSA disappeared in 31 (58.5%). Graft failure occurred in 16 patients at a median of 54 (range, 5-83) months, of whom 14 formed dnDSA. The 14 patients who lost their graft due to rejection, all had persistent C1q-dnDSA. C1q-binding status improved the individual risk assessment, with persistent; C1q binding yielding the strongest independent association of graft failure (hazard ratio, 45.5; 95% confidence interval, 11.7-177.4). C1q-dnDSA is more useful than standard dnDSA as a noninvasive biomarker for identifying patients at the highest risk of graft failure.

  View details for DOI 10.3389/ti.2021.10158

  View details for PubMedID 35992747

  View details for PubMedCentralID PMC9386741

• Next generation sequencing confirms T-cell clonality in a subset of pediatric pityriasis lichenoides. Journal of cutaneous pathology Raghavan, S. S., Wang, J. Y., Gru, A. A., Marqueling, A. L., Teng, J. M., Brown, R. A., Novoa, R. A., Kim, Y., Zehnder, J., Zhang, B. M., Rieger, K. E. 2021

  Abstract

  Pityriasis lichenoides (PL) is a papulosquamous disease that affects both adults and children. Previous studies have shown a subset of this entity to have clonal T-cell populations via PCR based assays. In this study, we sought to implement next generation sequencing as a more sensitive and specific test to examine for T-cell clonality within the pediatric population.We identified 18 biopsy specimens from 12 pediatric patients with clinical and histopathologic findings compatible with PL. Patient demographics, clinical features, management, and histopathologic findings were reviewed. All specimens were analyzed for clonality with next generation sequencing of T-cell receptor beta (TRB) and gamma (TRG) genes.Of the 12 patients, 9 (75%) had complete resolution of lesions at the time of data collection (mean follow up 31 months). The remaining three patients significantly improved with methotrexate (with or without acitretin). Interestingly, 7 of 12 patients (58%) and 9 of 17 biopsy specimens (53%) showed evidence of T-cell clonality. Two patients showed matching TRB clones from different anatomic sites.T-cell clonality is a common finding in PL, likely representing a "reactive clonality" rather than a true lymphoproliferative disorder. Clonality alone cannot be used as a means to distinguish PL from lymphomatoid papulosis or cutaneous lymphoma. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1111/cup.14143

  View details for PubMedID 34614220

• The Role of Donor KIR Genotyping in Pediatric Hematopoietic Stem Cell Transplantation For Non-Malignant Disorders Mariano, L., Bertaina, A., Zhang, B. M., Kristovich, K., Vina, M. SPRINGERNATURE. 2020: 640

  View details for Web of Science ID 000600556202248

• Chimerism Monitoring with Highly Sensitive and Precise Next-Generation Sequencing Assay in Patients Post-Allogeneic Hematopoietic Stem Cell Transplantation Sharma, D., Egidio, C., Grskovic, M., Vina, M., Zhang, B. M. ELSEVIER SCIENCE INC. 2020: S310

  View details for Web of Science ID 000516887900473

• Next Generation Sequencing-Based Characterization of T Cell Receptor Repertoire of Patients with Immune Thrombocytopenia Han, P., You, X., Lo, C., Xu, L., Zhang, H., Zehnder, J. L., Zhang, B. AMER SOC HEMATOLOGY. 2019

  View details for DOI 10.1182/blood-2019-127590

  View details for Web of Science ID 000577160405007

• Non-HLA Antibody-Mediated Rejection of Lung Transplant Masquerading Transfusion-Related Acute Lung Injury Yunce, M., Virk, M., Zhang, B. M., Mooney, K., Saleem, A., Shan, H. WILEY. 2019: 199A

  View details for Web of Science ID 000502826600470

• Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection. Molecular diagnosis & therapy Atkins, A., Gupta, P., Zhang, B. M., Tsai, W., Lucas, J., Javey, M., Vora, A., Mei, R. 2019

  Abstract

  INTRODUCTION: Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges.METHODS: The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm.RESULTS: We have demonstrated that OncoLBx detects VAFs of≥0.1% for SNVs and indels,≥0.5% for fusions,≥4.5 copies for CNVs and≥2% for MSI, with all variant types having specificity≥99.999%. Diagnostic performance of paired samples displays 80% sensitivity and>99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.

  View details for DOI 10.1007/s40291-019-00406-0

  View details for PubMedID 31209714

• Case series of MET exon 14 skipping mutation-positive non-small-cell lung cancers with response to crizotinib and cabozantinib ANTI-CANCER DRUGS Wang, S. Y., Zhang, B. M., Wakelee, H. A., Koontz, M. Z., Pan, M., Diehn, M., Kunder, C. A., Neal, J. W. 2019; 30 (5): 537–41

  View details for DOI 10.1097/CAD.0000000000000765

  View details for Web of Science ID 000466009600014

• De novo complement-activating donor-specific antibodies in pediatric renal transplant recipients are highly responsive to therapy Sigurjonsdottir, V., Zhang, B. M., Vina, M., Chaudhuri, A., Concepcion, W., Gallo, A., Grimm, P. WILEY. 2019

  View details for Web of Science ID 000485482200137

• Chimerism Analysis in Pediatric Hematopoietic Stem Cell Transplantation for Non-Malignant Disorders Mariano, L., Zhang, B., Kristovich, K., Agarwal-Hashmi, R., Roncarolo, M., Bertaina, A., Fernandez-Vina, M. ELSEVIER SCIENCE INC. 2019

  View details for Web of Science ID 000540655500467

• Case series of MET exon 14 skipping mutation-positive non-small-cell lung cancers with response to crizotinib and cabozantinib. Anti-cancer drugs Wang, S. X., Zhang, B. M., Wakelee, H. A., Koontz, M. Z., Pan, M., Diehn, M., Kunder, C. A., Neal, J. W. 2019

  Abstract

  The mesenchymal-to-epithelial transition (MET) gene is altered and becomes a driver mutation in up to 5% of non-small-cell lung cancer (NSCLC). We report our institutional experience treating patients with MET exon 14 skipping (METex14) mutations, including responses to the MET inhibitors crizotinib and cabozantinib. We identified cases of NSCLC with METex14 mutations using an institutionally developed or commercial next-generation sequencing assay. We assessed patient and disease characteristics by retrospective chart review. Some patients were treated off-label by the physician with crizotinib or cabozantinib, and tumor responses to these agents were assessed. A total of 15 patients with METex14-mutated NSCLC were identified, predominantly male (n=10) with a smoking history (60%) and a median age of 74.0 years. No other actionable somatic mutations were detected. Stage distribution included 26.7% stage I, 6.7% stage II, 6.7% stage III, and 60.0% stage IV. Among patients treated with crizotinib or cabozantinib (n=6), three patients showed partial response and one patient showed stable disease on the basis of RECIST criteria. Four patients experienced side effects requiring drug holiday, reduction, or cessation. Our findings highlight the diversity in presentation and histology of NSCLC with METex14 mutations, which were found in the absence of other actionable driver mutations. We observed evidence of tumor response to crizotinib and cabozantinib, supporting the previous reports that METex14 mutations in NSCLC are actionable driver events.

  View details for PubMedID 30762593

• Blood transcriptome and clonal T cell correlates of response and nonresponse to eltrombopag therapy in a cohort of patients with chronic immune thrombocytopenia. Haematologica Zhang, H. n., Zhang, B. M., Guo, X. n., Xu, L. n., You, X. n., West, R. B., Bussel, J. B., Zehnder, J. L. 2019

  View details for DOI 10.3324/haematol.2019.226688

  View details for PubMedID 31296576

• Molecular Diagnosis in Hematology Wintrobe’s Clinical Hematology Zhang, B. M., Zehnder, J. L. 2019; 14e: 56–66
• Assessment by Extended-Coverage Next-Generation Sequencing Typing of DPA1 and DPB1 Mismatches in Siblings Matching at HLA-A, -B, -C, -DRB1, and -DQ Loci. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation Mariano, L. n., Zhang, B. M., Osoegawa, K. n., Lowsky, R. n., Fernandez-Vina, M. n. 2019

  Abstract

  Allogeneic hematopoietic stem cell transplant from an HLA matched sibling donor is usually the preferable choice. The use of next-generation sequencing (NGS) for HLA typing in clinical practice provides broader coverage and higher resolution of HLA genes. We evaluated the frequency of DPB1 crossing-over events among patients and potential related donors typed with NGS. From July 2016 to January 2018, 593 patients and 2385 siblings were typed. We evaluated sibling matching status in 546 patients, and 44.8% of these patients had siblings that matched at HLA-A, -B, -C, -DRB1, and -DQB1 loci. In 306 patient-HLA matched sibling pairs, we found 6 pairs (1.96%) with 1 DPB1 mismatch, and 5 of these pairs included an additional mismatch in DPA1. No additional mismatches were observed at the low expression loci. Using the T cell epitope algorithm, 4 of these DP mismatches were classified as permissive, 1 as nonpermissive in the host-versus-graft direction, and 1 as nonpermissive in the graft-versus-host direction. The frequency of DPB1 and DPA1 mismatches is low, and their impact in related donor transplants is not well established. Although DP typing in related transplants goes beyond guidelines, it is especially relevant for sensitized patients. NGS-based HLA typing provides full gene coverage, and its use in clinical practice can enable better donor selection.

  View details for DOI 10.1016/j.bbmt.2019.07.033

  View details for PubMedID 31381995

• Case Series of MET Exon 14 Skipping Mutation-positive Non-Small Cell Lung Cancers and Response to Crizotinib. International journal of radiation oncology, biology, physics Wang, S. X., Zhang, B., Wakelee, H. A., Diehn, M., Kunder, C., Neal, J. W. 2017; 98 (1): 239-?

  View details for DOI 10.1016/j.ijrobp.2017.01.170

  View details for PubMedID 28587017

• Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use JOURNAL OF MOLECULAR DIAGNOSTICS Xu, L., You, X., Zheng, P., Zhang, B. M., Gupta, P. K., Lavori, P., Meyer, E., Zehnder, J. L. 2017; 19 (1): 72-83

  Abstract

  Next-generation sequencing (NGS) of immune receptors has become a standard tool to assess minimal residual disease (MRD) in patients treated for lymphoid malignancy, and it is being used to study the T-cell repertoire in many clinical settings. To better understanding the potential clinical utility and limitations of this application outside of MRD, we developed a BIOMED-2 primer-based NGS method and characterized its performance in controls and patients with graft-versus-host disease (GVHD) after allogeneic hematopoietic transplant. For controls and patients with GVHD, replicate sequencing of the same T-cell receptor β (TRB) libraries was highly reproducible. Higher variability was observed in sequencing of different TRB libraries made from the same DNA stock. Variability was increased in patients with GVHD compared with controls; patients with GVHD also had lower diversity than controls. In the T-cell repertoire of a healthy person, approximately 99.6% of the CDR3 clones were in low abundance, with frequency <10(-3). A single library could identify >93% of the clones with frequency ≥10(-3) in the repertoire. Sequencing in duplicate increased the average detection rate to >97%. This work demonstrates that NGS reliably and robustly characterizes TRB populations in healthy individuals and patients with GVHD with frequency ≥10(-3) and provides a methodologic framework for applying NGS immune repertoire methods to clinical testing applications beyond MRD.

  View details for DOI 10.1016/j.jmoldx.2016.07.009

  View details for Web of Science ID 000390983100008

• Identification of a Novel MPL Loss of Function Mutation in a Patient with Cyclic Thrombocytopenia and Characterization of This Syndrome Zhang, H., Hou, Y., Brar, R. S., Zhang, B., Chen, Z., Abidi, P., Jin, J., Gotlib, J. R., Zehnder, J. L. AMER SOC HEMATOLOGY. 2016

  View details for Web of Science ID 000394452300114

• PS01.67: Case Series of MET Exon 14 Skipping Mutation-Positive Non-Small Cell Lung Cancers and Response to Crizotinib: Topic: Medical Oncology. Journal of thoracic oncology Wang, S. X., Zhang, B. M., Wakelee, H., Diehn, M., Kunder, C. A., Neal, J. W. 2016; 11 (11S): S312-S313

  View details for DOI 10.1016/j.jtho.2016.09.102

  View details for PubMedID 27969534

• ROS: novel regulators of thrombopoiesis BLOOD Zhang, B., Zehnder, J. L. 2016; 128 (5): 613-?

  View details for DOI 10.1182/blood-2016-06-718544

  View details for Web of Science ID 000383843000004

  View details for PubMedID 27492312

• Effects of Thrombopoietin Mimetics on Patients with Chronic ITP: Perspectives from Blood Transcriptome Profiling Analysis Zhang, H., Zhang, B., Guo, X., Haq, N., West, R. B., Bussel, J. B., Zehnder, J. L. AMER SOC HEMATOLOGY. 2014

  View details for Web of Science ID 000349233800107

• Immunoglobulin and T cell receptor gene high-throughput sequencing quantifies minimal residual disease in acute lymphoblastic leukemia and predicts post-transplantation relapse and survival. Biology of blood and marrow transplantation Logan, A. C., Vashi, N., Faham, M., Carlton, V., Kong, K., Buño, I., Zheng, J., Moorhead, M., Klinger, M., Zhang, B., Waqar, A., Zehnder, J. L., Miklos, D. B. 2014; 20 (9): 1307-1313

  Abstract

  Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10(-4) after induction predicts subsequent relapse. Likewise, MRD ≥ 10(-4) in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10(-6) or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10(-4) before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10(-6) had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.

  View details for DOI 10.1016/j.bbmt.2014.04.018

  View details for PubMedID 24769317

• Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes. Haematologica Merker, J. D., Roskin, K. M., Ng, D., Pan, C., Fisk, D. G., King, J. J., Hoh, R., Stadler, M., Okumoto, L. M., Abidi, P., Hewitt, R., Jones, C. D., Gojenola, L., Clark, M. J., Zhang, B., Cherry, A. M., George, T. I., Snyder, M., Boyd, S. D., Zehnder, J. L., Fire, A. Z., Gotlib, J. 2013; 98 (11): 1689-1696

  Abstract

  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

• Minimal residual disease quantification using consensus primers and high- throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia LEUKEMIA Logan, A. C., Zhang, B., Narasimhan, B., Carlton, V., Zheng, J., Moorhead, M., Krampf, M. R., Jones, C. D., Waqar, A. N., Faham, M., Zehnder, J. L., Miklos, D. B. 2013; 27 (8): 1659-1665

  Abstract

  Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH-HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10(-4) and 20% in those with MRD 10(-4) (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5-32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden 10(-5) was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH-HTS method may facilitate routine MRD quantification in clinical trials.Leukemia advance online publication, 12 March 2013; doi:10.1038/leu.2013.52.

  View details for DOI 10.1038/leu.2013.52

  View details for Web of Science ID 000322823200006

  View details for PubMedID 23419792

• Oxidative stress and immune thrombocytopenia. Seminars in hematology Zhang, B., Zehnder, J. L. 2013; 50 (3): e1-4

  Abstract

  Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by increased platelet destruction or decreased platelet production. The mechanism of the disease has been extensively studied so that we now have a much improved understanding of the pathophysiology; however, the trigger of the autoimmunity remains unclear. More recently, oxidative stress was identified to be involved in the pathogenesis of ITP and provides a new hypothesis for the initiation of autoimmunity in patients with ITP. In this review, oxidative stress and its impact on autoimmunity, particularly ITP, will be covered.

  View details for DOI 10.1053/j.seminhematol.2013.06.011

  View details for PubMedID 23953344

• Desktop transcriptome sequencing from archival tissue to identify clinically relevant translocations. American journal of surgical pathology Sweeney, R. T., Zhang, B., Zhu, S. X., Varma, S., Smith, K. S., Montgomery, S. B., van de Rijn, M., Zehnder, J., West, R. B. 2013; 37 (6): 796-803

  Abstract

  Somatic mutations, often translocations or single nucleotide variations, are pathognomonic for certain types of cancers and are increasingly of clinical importance for diagnosis and prediction of response to therapy. Conventional clinical assays only evaluate 1 mutation at a time, and targeted tests are often constrained to identify only the most common mutations. Genome-wide or transcriptome-wide high-throughput sequencing (HTS) of clinical samples offers an opportunity to evaluate for all clinically significant mutations with a single test. Recently a "desktop version" of HTS has become available, but most of the experience to date is based on data obtained from high-quality DNA from frozen specimens. In this study, we demonstrate, as a proof of principle, that translocations in sarcomas can be diagnosed from formalin-fixed paraffin-embedded (FFPE) tissue with desktop HTS. Using the first generation MiSeq platform, full transcriptome sequencing was performed on FFPE material from archival blocks of 3 synovial sarcomas, 3 myxoid liposarcomas, 2 Ewing sarcomas, and 1 clear cell sarcoma. Mapping the reads to the "sarcomatome" (all known 83 genes involved in translocations and mutations in sarcoma) and using a novel algorithm for ranking fusion candidates, the pathognomonic fusions and the exact breakpoints were identified in all cases of synovial sarcoma, myxoid liposarcoma, and clear cell sarcoma. The Ewing sarcoma fusion gene was detectable in FFPE material only with a sequencing platform that generates greater sequencing depth. The results show that a single transcriptome HTS assay, from FFPE, has the potential to replace conventional molecular diagnostic techniques for the evaluation of clinically relevant mutations in cancer.

  View details for DOI 10.1097/PAS.0b013e31827ad9b2

  View details for PubMedID 23598961

• Sequential azacitidine plus lenalidomide combination for elderly patients with untreated acute myeloid leukemia. Haematologica Pollyea, D. A., Zehnder, J., Coutre, S., Gotlib, J. R., Gallegos, L., Abdel-Wahab, O., Greenberg, P., Zhang, B., Liedtke, M., Berube, C., Levine, R., Mitchell, B. S., Medeiros, B. C. 2013; 98 (4): 591-596

  Abstract

  There are limited treatment options for older patients with acute myeloid leukemia and prognosis of these patients remains poor, thereby warranting development of novel therapies. We evaluated the efficacy and safety of azacitidine in combination with lenalidomide as front-line therapy for older patients with acute myeloid leukemia. Patients ≥ 60 years of age with untreated acute myeloid leukemia received azacitidine 75 mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 days starting on day 8 of each cycle every 6 weeks. Patients received continued therapy until disease progression, unacceptable toxicity, or completion of 12 cycles. Forty-two patients (median age, 74 years) were enrolled with equal distribution according to European LeukemiaNet risk. The overall response rate was 40% (rate of complete remission with or without complete recovery of blood counts = 28%). The median time to complete remission with or without complete recovery of blood counts was 12 weeks, and duration of this status was 28 weeks (range, 4 - >104 weeks). Therapy-related acute myeloid leukemia and a high score on the Hematopoietic Cell Transplantation Comorbidity Index were negative predictors of response. Early death was noted in 17% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosuppression. In conclusion, a sequential combination of azacitidine plus lenalidomide has clinical activity in older patients with acute myeloid leukemia, and further studies of this combination are underway.

  View details for DOI 10.3324/haematol.2012.076414

  View details for PubMedID 23242596

• 2-Hydroxyglutarate in IDH mutant acute myeloid leukemia: predicting patient responses, minimal residual disease and correlations with methylcytosine and hydroxymethylcytosine levels LEUKEMIA & LYMPHOMA Pollyea, D. A., Kohrt, H. E., Zhang, B., Zehnder, J., Schenkein, D., Fantin, V., Straley, K., Vasanthakumar, A., Abdel-Wahab, O., Levine, R., Godley, L. A., Medeiros, B. C. 2013; 54 (2): 408-410

  View details for DOI 10.3109/10428194.2012.701009

  View details for Web of Science ID 000313285400034

  View details for PubMedID 22680765

• Impaired B Cell Clonotype Diversification After Allogeneic Hematopoietic Cell Transplantation Predicts Graft-Versus-Host Disease BMT Tandem Meetings Logan, A., Sahaf, B., Zhang, B., Arai, S., Carlton, V., Zheng, J., Moorhead, M., Krampf, M. R., Jones, C. D., Waqar, A. N., Faham, M., Shizuru, J. A., Zehnder, J. L., Miklos, D. B. ELSEVIER SCIENCE INC. 2013: S148–S149

  View details for Web of Science ID 000314441900072

• Whole Genome Sequence Analysis of Primary Myelofibrosis. 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Merker, J. D., Roskin, K., Ng, D., Pan, C., Fisk, D. G., Jones, C. D., Gojenola, L., Clark, M. J., Zhang, B., Cherry, M., Snyder, M., Boyd, S. D., Zehnder, J. L., Fire, A. Z., Gotlib, J. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000313838905376

• Azacitidine Plus Lenalidomide for Untreated AML Patients Ineligible for Conventional Chemotherapy Pollyea, D. A., Zehnder, J. L., Coutre, S., Gotlib, J., Gallegos, L., Greenberg, P., Zhang, B., Liedtke, M., Levine, R. L., Medeiros, B. C. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000313838906082

• The Role of Oxidative Stress in Pediatric Immune Thrombocytopenia Lo, C., Zhang, B., Cusmano-Ozog, K., Wong, W., Jeng, M., Cowan, T., Zehnder, J. L. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000314049602365

• Safety, efficacy and biological predictors of response to sequential azacitidine and lenalidomide for elderly patients with acute myeloid leukemia LEUKEMIA Pollyea, D. A., Kohrt, H. E., Gallegos, L., Figueroa, M. E., Abdel-Wahab, O., Zhang, B., Bhattacharya, S., Zehnder, J., Liedtke, M., Gotlib, J. R., Coutre, S., Berube, C., Melnick, A., Levine, R., Mitchell, B. S., Medeiros, B. C. 2012; 26 (5): 893-901

  Abstract

  Acute myeloid leukemia (AML) is a disease of the elderly. Poor outcomes with standard therapies necessitate novel approaches. Outpatient regimens sufficiently potent and well tolerated to induce remissions and enable continuation therapy may be beneficial. In this phase-1 study, we determined the maximum tolerated dose (MTD) and the efficacy for sequential azacitidine and lenalidomide as remission induction and continuation therapy in elderly, previously untreated patients. We investigated the impact on global DNA methylation and bone marrow cytokines, and sought biological predictors of response. Eighteen patients were enrolled. The MTD was not reached. Median follow-up was 8.2 months (10.3 months for survivors). Common adverse events included fatigue, injection site reactions, constipation, nausea, pruritus and febrile neutropenia. Ten patients responded (56%), and the rate of complete remissions (CRs) or CRs with incomplete recovery of blood counts for evaluable patients was 44% (7/16). The median response duration was 6.2 months. DNA demethylation and changes in bone marrow cytokines were observed; responders had a unique cytokine profile and a trend towards lower methylation levels. Sequential azacitidine and lenalidomide was well tolerated with encouraging clinical and biological activity in previously untreated elderly AML patients. This trial is registered at ClinicalTrials.gov (NCT00890929).

  View details for DOI 10.1038/leu.2011.294

  View details for Web of Science ID 000303883500005

  View details for PubMedID 22033493

• Tailored temozolomide therapy according to MGMT methylation status for elderly patients with acute myeloid leukemia AMERICAN JOURNAL OF HEMATOLOGY Medeiros, B. C., Kohrt, H. E., Gotlib, J., Coutre, S. E., Zhang, B., Arber, D. A., Zehnder, J. L. 2012; 87 (1): 45-50

  Abstract

  Temozolomide sensitivity is determined by methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter. This study assessed whether the temozolomide dose can be tailored by MGMT promoter status and whether protracted, low-dose temozolomide can "prime" blasts in patients with unmethylated MGMT (unMGMT). Elderly patients with high-risk AML were stratified by MGMT methylation. Patients with methylated MGMT (mMGMT) received temozolomide 200 mg/m(2) orally for 7 days every 4 weeks, while patients with unMGMT received temozolomide 100 mg/m(2) orally for 14 days followed by 200 mg/m(2) orally for 7 days every 6weeks. Of 36 patients (median age, 75 years), 31 (86%) had an unMGMT promoter. Overall response rate for the entire cohort was 36%. Patients with mMGMT and unMGMT had similar response rates (40% vs. 29%). Median duration of response and overall survival (OS) among responders were 29 and 35 weeks, respectively. Induction deaths (ID) occurred in 25% of patients, mostly caused by disease progression. Hematological toxicities were the most common adverse event. Toxicities were similar between patients on conventional versus protracted schedules. High HCT-CI scores were predictive of lower CR rate, higher ID, and shorter OS, while bone marrow blast count <50% at screening predicted for improved responses. Temozolomide, dosed according to MGMT methylation status, demonstrated modest clinical activity in elderly patients with AML, especially in those presenting with fewer comorbidities and low disease burden. The trial was registered on www.ClinicalTrials.gov as #NCT00611247.

  View details for DOI 10.1002/ajh.22191

  View details for Web of Science ID 000298257700010

  View details for PubMedID 22052619

• A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia BLOOD Ng, D., Jones, C., Oh, S. T., Nolan, G. P., Salehi, S., Wong, W., Zehnder, J. L., Gotlib, J. 2011; 118 (26): 6988-?

  View details for DOI 10.1182/blood-2011-10-386177

  View details for Web of Science ID 000298401000038

• 2-Hydroxyglutarate in IDH mutant AML: Predicting Patient Responses, Minimal Residual Disease and Correlations with Methylcytosine and Hydroxymethylcytosine Levels Pollyea, D. A., Kohrt, H. E., Zhang, B., Zehnder, J. L., Schenkein, D. P., Fantin, V., Straley, K., Vasanthakumar, A., Abdel-Wahab, O., Levine, R. L., Godley, L., Medeiros, B. C. AMER SOC HEMATOLOGY. 2011: 1073–74

  View details for Web of Science ID 000299597103375

• Identification of a Novel Splice Donor Mutation In the Thrombopoietin Gene In a Philippine Family with Hereditary Thrombocythemia 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Gotlib, J., Zhang, B., Jones, C. D., Riess, J., Wong, W. B., Simonds, E. F., Hale, M. B., Abidi, P., McClung, J., Nolan, G. P., Oh, S. T., Zehnder, J. L. AMER SOC HEMATOLOGY. 2010: 1272–72

  View details for Web of Science ID 000289662203418

• Identification of Novel LNK Mutations In Patients with Chronic Myeloproliferative Neoplasms and Related Disorders 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Oh, S. T., Zahn, J. M., Jones, C. D., Zhang, B., Loh, M. L., Kantarjian, H., Simonds, E. F., Bruggner, R. V., Abidi, P., Natsoulis, G., Bell, J., Buenrostro, J., Nolan, G. P., Zehnder, J. L., Ji, H. P., Gotlib, J. AMER SOC HEMATOLOGY. 2010: 143–44

  View details for Web of Science ID 000289662200316

• Combined Use of PCR-Based TCRG and TCRB Clonality Tests on Paraffin-Embedded Skin Tissue in the Differential Diagnosis of Mycosis Fungoides and Inflammatory Dermatoses JOURNAL OF MOLECULAR DIAGNOSTICS Zhang, B., Beck, A. H., Taube, J. M., Kohler, S., Seo, K., Zwerner, J., Viakhereva, N., Sundram, U., Kim, Y. H., Schrijver, I., Arber, D. A., Zehnder, J. L. 2010; 12 (3): 320-327

  Abstract

  The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.

  View details for DOI 10.2353/jmoldx.2010.090123

  View details for Web of Science ID 000277531700009

  View details for PubMedID 20203005

  View details for PubMedCentralID PMC2860468

• Increased VNN1/PPARG Gene Expression Ratio Is Correlated with Developing Chronic ITP and Oxidative Stress Exposure to PBMC in Vitro 51st Annual Meeting and Exposition of the American-Society-of-Hematology Zhang, B., Shen, L., Jeng, M., Jones, C., Wong, W., Engleman, E. E., Zehnder, J. L. AMER SOC HEMATOLOGY. 2009: 368–68

  View details for Web of Science ID 000272725801073

• Elevated Vanin 1 and Advillin Expression Is Associated with Progression to Chronic ITP in Children 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Zhang, B., Sood, R., Jones, C., Wong, W., Jeng, M., Zehnder, J. L. AMER SOC HEMATOLOGY. 2008: 152–53

  View details for Web of Science ID 000262104700398

• Large granular lymphocyte leukemia: Clonality reconsidered. Witteles, W., Zhang, B., Schrijver, I., Arber, D., Gotlib, J., Zehnder, J. AMER SOC HEMATOLOGY. 2007: 912A

  View details for Web of Science ID 000251100804139

• T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides J Am Acad Dermatol. Thurber, S. E., Zhang, B., Kim, Y. H., Schrijver, I., Zehnder, J. L., Kohler, S. 2007; 57 (5)