Publications by authors named "James L Zehnder"

110 Publications

Incidence and Risk Factors Associated with Bleeding and Thrombosis Following Chimeric Antigen Receptor T Cell Therapy.

Blood Adv 2021 Sep 14. Epub 2021 Sep 14.

Stanford University, Stanford, California, United States.

Bleeding and thrombotic events are an emerging toxicity associated with chimeric antigen receptor (CAR) therapies. To determine their incidence, we retrospectively analyzed consecutive adult patients (n=127) with large B-cell lymphoma (LBCL) or B-cell acute lymphoblastic leukemia (B-ALL) treated between 2017-2020 with axicabtagene ciloleucel (axi-cel) (N=89) or a bispecific CD19/CD22 CAR (N=38). 12 (9.4%) and 8 (6.3%) patients developed bleeding and thrombosis within first 3 months, respectively. In the axi-cel subgroup, these occurred in 11.2% and 6.7%, respectively. Bleeding occurred between days 8-30 (median 17.5), and thrombosis between days 2-91 (median 29). Bleeding sites included genitourinary (N=6), soft tissue (N=2), intracranial (N=2), gastrointestinal (N=1), pulmonary (N=1), and were associated with features of consumptive coagulopathy. On univariate analysis, patients with bleeding were older (median 72 vs. 60 yrs, P<0.01), had lower baseline platelets (86 vs. 178 K/uL, P<0.01), lower platelet nadir after CAR-T (median 17.5 vs. 48 K/uL; P<0.01), lower fibrinogen nadir (median 122 vs. 340 ug/mL; P<0.01) and elevated LDH (P=0.01). ICANS grade ≥3 was associated with increased bleeding (50% vs. 15%; P=0.01), thrombosis (50% vs. 16%; P=0.04), PT prolongation, hypofibrinogenemia and elevated D-dimer. A paucity of events limited multivariate analysis, however low pre-treatment platelets were associated with bleeding in a multivariate logistic regression model. Patients with thrombocytopenia or severe ICANS are at increased risk of bleeding complications and should be closely monitored particularly within the first month after CAR therapy. Future studies in larger cohorts should assess risk factors for systemic coagulopathies in CAR-T therapy, including their association with neurotoxicity.
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http://dx.doi.org/10.1182/bloodadvances.2021004716DOI Listing
September 2021

Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

J Mol Diagn 2021 Aug 5. Epub 2021 Aug 5.

Department of Pathology, Stanford University School of Medicine, Stanford, California. Electronic address:

FLT3 internal tandem duplications (ITDs) are found in approximately one-third of patients with acute myeloid leukemia and have important prognostic and therapeutic implications that have supported their assessment in routine clinical practice. Conventional methods for assessing FLT3-ITD status and allele burden have been primarily limited to PCR fragment size analysis because of the inherent difficulty in detecting large ITD variants by next-generation sequencing (NGS). In this study, we assess the performance of publicly available bioinformatic tools for the detection and quantification of FLT3-ITDs in clinical hybridization-capture NGS data. We found that FLT3_ITD_ext had the highest overall accuracy for detecting FLT3-ITDs and was able to accurately quantify allele burden. Although all other tools evaluated were able to detect FLT3-ITDs reasonably well, allele burden was consistently underestimated. We were able to significantly improve quantification of FLT3-ITD allelic burden independent of the detection method by utilizing soft-clipped reads and/or ITD junctional sequences. In addition, we show that identifying mutant reads by previously identified junctional sequences further improves the sensitivity of detecting FLT3-ITDs in post-treatment samples. Our results demonstrate that FLT3-ITDs can be reliably detected in clinical NGS data using available bioinformatic tools. We further describe how accurate quantification of FLT3-ITD allele burden can be added on to existing clinical NGS pipelines for routine assessment of FLT3-ITD status in patients with acute myeloid leukemia.
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http://dx.doi.org/10.1016/j.jmoldx.2021.07.012DOI Listing
August 2021

Validation of a Next-Generation Sequencing-Based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing.

J Mol Diagn 2021 07 21;23(7):805-815. Epub 2021 Apr 21.

Department of Pathology, Stanford University School of Medicine, Stanford, California. Electronic address:

Assessment of T-cell receptor γ gene (TRG) rearrangements is an importants consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard of 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, given that they generate results with specific clonotype sequence information and allow for more accurate quantitation. The present study evaluated various test parameters and performance characteristics of 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%. The concordance rates were 88% to 95% in the V1-8, V10, and V11 gene families, but lower in the V9 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 clonotypes of the same fragment size. This 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.
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http://dx.doi.org/10.1016/j.jmoldx.2021.03.008DOI Listing
July 2021

DOAC-Stop in lupus anticoagulant testing: Direct oral anticoagulant interference removed in most samples.

Res Pract Thromb Haemost 2021 Feb 27;5(2):314-325. Epub 2021 Jan 27.

Department of Pathology Stanford University School of Medicine Stanford CA USA.

Background: The use of direct oral anticoagulants (DOACs) is a convenient therapeutic option for patients at risk of thrombosis. DOACs interfere with clot-based testing for the identification of lupus anticoagulant antibodies (LACs) in patients with antiphospholipid syndrome (APS), a common cause of acquired thrombotic disease.

Objectives: To evaluate a commercially available reagent DOAC-Stop for the removal of DOAC interference encountered in LAC testing.

Patients/methods: We collected a cohort of 73 test samples from patients on DOAC therapy identified at a large institutional coagulation laboratory from March to December 2019, along with samples from 40 LAC positive and negative control patients not on therapy. Samples were treated with DOAC-Stop and tested for anti-Xa activity and thrombin time for the removal of apixaban, rivaroxaban, argatroban, and dabigatran activity from patient samples. Treated and untreated samples were tested using the activated partial thromboplastin time, silica clotting time, and dilute Russell's viper venom time to evaluate the reliability and utility of DOAC-Stop.

Results: DOAC-Stop markedly reduced DOAC interference from test samples ( < .05). DOAC-Stop had no effect on LAC testing in the absence of DOAC therapy, permitting the identification of all LAC positive and negative controls. DOAC-Stop removed false positives and false negatives resulting from DOAC interference and allows the identification of patients meeting criteria for the diagnosis of APS by LAC testing, as well as the detection of patients on rivaroxaban who are triple positive for APS.

Conclusions: DOAC-Stop is an effective adjunct for the clinical laboratory faced with DOAC interference in LAC testing.
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http://dx.doi.org/10.1002/rth2.12472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938630PMC
February 2021

Mapping of SARS-CoV-2 Brain Invasion and Histopathology in COVID-19 Disease.

medRxiv 2021 Feb 18. Epub 2021 Feb 18.

The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal β-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.
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http://dx.doi.org/10.1101/2021.02.15.21251511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899461PMC
February 2021

Identification of a pathogenic variant in a Chinese family with congenital macrothrombocytopenia through whole genome sequencing.

Platelets 2021 Jan 5:1-5. Epub 2021 Jan 5.

Department of Hematology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.

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 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 -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.
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http://dx.doi.org/10.1080/09537104.2020.1869714DOI Listing
January 2021

A Blueprint for Identifying Phenotypes and Drug Targets in Complex Disorders with Empirical Dynamics.

Patterns (N Y) 2020 Dec 6;1(9):100138. Epub 2020 Nov 6.

Département de Mathématiques et de Statistique, Université de Montréal, Montréal, QC, Canada.

A central challenge in medicine is translating from observational understanding to mechanistic understanding, where some observations are recognized as causes for the others. This can lead not only to new treatments and understanding, but also to recognition of novel phenotypes. Here, we apply a collection of mathematical techniques (), which infer mechanistic networks in a model-free manner from longitudinal data, to hematopoiesis. Our study consists of three subjects with markers for cyclic thrombocytopenia, in which multiple cells and proteins undergo abnormal oscillations. One subject has atypical markers and may represent a rare phenotype. Our analyses support this contention, and also lend new evidence to a theory for the cause of this disorder. Simulations of an intervention yield encouraging results, even when applied to patient data outside our three subjects. These successes suggest that this blueprint has broader applicability in understanding and treating complex disorders.
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http://dx.doi.org/10.1016/j.patter.2020.100138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733879PMC
December 2020

Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome.

Sci Immunol 2020 12;5(54)

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.
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http://dx.doi.org/10.1126/sciimmunol.abe0240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857392PMC
December 2020

Cutaneous T-cell lymphomas with pathogenic somatic mutations and absence of detectable clonal T-cell receptor gene rearrangement: two case reports.

Diagn Pathol 2020 Sep 28;15(1):122. Epub 2020 Sep 28.

Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA.

Background: Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of extranodal non-Hodgkin lymphomas for which diagnosis can be challenging given the potential for overlap with inflammatory dermatoses. Current diagnostic criteria for CTCL incorporate clinical and histopathologic findings as well as results of T-cell receptor (TCR) gene sequencing. Molecular interrogation of TCR genes, TRG and TRB, has proven to be a critical tool for confirming diagnoses of CTCL and for disease tracking after initiation of therapy or after stem cell transplant. Methods for confirming a diagnosis of lymphoma in the absence of TCR gene clonality are lacking. We present two patients with CTCL with pathogenic somatic mutations in the absence of TRG and TRB clonality.

Case Presentations: Case 1: A 38-year-old male had a 19-year history of a diffuse skin rash with papulosquamous, granulomatous, and verrucous features and progressive ulcerated plaques and tumors demonstrating an atypical CD4+ T-cell infiltrate with expression of cytotoxic markers CD56, TIA-1, granzyme, and perforin on histopathology. No definitive evidence for T-cell clonality was detected by conventional PCR of 6 biopsies or by next-generation sequencing (NGS) of 14 biopsies. Somatic mutational profiling of a skin biopsy revealed pathogenic mutations in PIKC3D and TERT promoter hotspots, confirming the presence of a clonal process. Case 2: A 69-year-old male with a 13-year history of progressive, diffuse hypertrophic and eroded plaques showed an atypical CD4+ T-cell infiltrate with subset expression of TIA-1 and granzyme on histopathology. No TCR clonality was detected by TCR-NGS of 6 biopsies. Somatic mutational profiling of a skin biopsy detected a pathogenic mutation in TP53, confirming the presence of a clonal process.

Conclusions: These cases highlight how detection of pathogenic somatic mutations can confirm a diagnosis of lymphoma in a clinically and histopathologically suspicious cutaneous lymphoid proliferation without detectable TCR clonality.
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http://dx.doi.org/10.1186/s13000-020-01022-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523289PMC
September 2020

Recipient-specific T-cell repertoire reconstitution in the gut following murine hematopoietic cell transplant.

Blood Adv 2020 09;4(17):4232-4243

Blood and Marrow Transplantation.

Graft-versus-host disease (GVHD) is a complication of hematopoietic cell transplantation (HCT) caused by alloreactive T cells. Murine models of HCT are used to understand GVHD and T-cell reconstitution in GVHD target organs, most notably the gastrointestinal (GI) tract where the disease contributes most to patient mortality. T-cell receptor (TCR) repertoire sequencing was used to measure T-cell reconstitution from the same donor graft (C57BL/6 H-2b) in the GI tract of different recipients across a spectrum of matching, from syngeneic (C57BL/6), to minor histocompatibility (MHC) antigen mismatch BALB.B (H-2b), to major MHC mismatched B10.BR (H-2k) and BALB/c (H-2d). Although the donor T-cell pools had highly similar TCR, the TCR repertoire after HCT was very specific to recipients in each experiment independent of geography. A single invariant natural killer T clone was identifiable in every recipient group and was enriched in syngeneic recipients according to clonal count and confirmatory flow cytometry. Using a novel cluster analysis of the TCR repertoire, we could classify recipient groups based only on their CDR3 size distribution or TCR repertoire relatedness. Using a method for evaluating the contribution of common TCR motifs to relatedness, we found that reproducible sets of clones were associated with specific recipient groups within each experiment and that relatedness did not necessarily depend on the most common clones in allogeneic recipients. This finding suggests that TCR reconstitution is highly stochastic and likely does not depend on the evaluation of the most expanded TCR clones in any individual recipient but instead depends on a complex polyclonal architecture.
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http://dx.doi.org/10.1182/bloodadvances.2019000977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479954PMC
September 2020

SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness.

medRxiv 2020 Aug 17. Epub 2020 Aug 17.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.
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http://dx.doi.org/10.1101/2020.08.15.20175794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444305PMC
August 2020

Ex vivo drug screening defines novel drug sensitivity patterns for informing personalized therapy in myeloid neoplasms.

Blood Adv 2020 06;4(12):2768-2778

Division of Hematology, Department of Medicine, Stanford University Medical Center, Stanford, CA; and.

Precision medicine approaches such as ex vivo drug sensitivity screening (DSS) are appealing to inform rational drug selection in myelodysplastic syndromes (MDSs) and acute myeloid leukemia, given their marked biologic heterogeneity. We evaluated a novel, fully automated ex vivo DSS platform that uses high-throughput flow cytometry in 54 patients with newly diagnosed or treatment-refractory myeloid neoplasms to evaluate sensitivity (blast cytotoxicity and differentiation) to 74 US Food and Drug Administration-approved or investigational drugs and 36 drug combinations. After piloting the platform in 33 patients, we conducted a prospective feasibility study enrolling 21 patients refractory to hypomethylating agents (HMAs) to determine whether this assay could be performed within a clinically actionable time frame and could accurately predict clinical responses in vivo. When assayed for cytotoxicity, ex vivo drug sensitivity patterns were heterogeneous, but they defined distinct patient clusters with differential sensitivity to HMAs, anthracyclines, histone deacetylase inhibitors, and kinase inhibitors (P < .001 among clusters) and demonstrated synergy between HMAs and venetoclax (P < .01 for combinations vs single agents). In our feasibility study, ex vivo DSS results were available at a median of 15 days after bone marrow biopsy, and they informed personalized therapy, which frequently included venetoclax combinations, kinase inhibitors, differentiative agents, and androgens. In 21 patients with available ex vivo and in vivo clinical response data, the DSS platform had a positive predictive value of 0.92, negative predictive value of 0.82, and overall accuracy of 0.85. These data demonstrate the utility of this approach for identifying potentially useful and often novel therapeutic drugs for patients with myeloid neoplasms refractory to standard therapies.
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http://dx.doi.org/10.1182/bloodadvances.2020001934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322964PMC
June 2020

Is Merkel Cell Carcinoma of Lymph Node Actually Metastatic Cutaneous Merkel Cell Carcinoma?

Am J Clin Pathol 2020 08;154(3):369-380

Department of Pathology, Stanford University School of Medicine, Stanford, CA.

Objectives: The possibility of a so-called primary lymph node neuroendocrine carcinoma has been described in the literature. Here we evaluate cases fitting such a diagnosis and find that the cases demonstrate a convincing and pervasive pattern consistent with metastatic Merkel cell carcinoma.

Methods: Six cases of primary lymph node Merkel cell carcinoma and one case of metastatic neuroendocrine carcinoma at a bony site, all with unknown primary, were sequenced using a combination of whole-exome and targeted panel methods. Sequencing results were analyzed for the presence of an ultraviolet (UV) mutational signature or off-target detection of Merkel cell polyomavirus (MCPyV).

Results: Four of six primary lymph node cases were positive for a UV mutational signature, with the remaining two cases positive for off-target alignment of MCPyV. One case of neuroendocrine carcinoma occurring at a bony site was also positive for a UV mutational signature.

Conclusions: We find no evidence to corroborate the existence of so-called primary Merkel cell carcinoma of lymph node.
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http://dx.doi.org/10.1093/ajcp/aqaa051DOI Listing
August 2020

Increasing Clinical Trial Accrual via Automated Matching of Biomarker Criteria.

Pac Symp Biocomput 2020 ;25:31-42

Departments of Biomedical Data Science and of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA,

Successful implementation of precision oncology requires both the deployment of nucleic acid sequencing panels to identify clinically actionable biomarkers, and the efficient screening of patient biomarker eligibility to on-going clinical trials and therapies. This process is typically performed manually by biocurators, geneticists, pathologists, and oncologists; however, this is a time-intensive, and inconsistent process amongst healthcare providers. We present the development of a feature matching algorithmic pipeline that identifies patients who meet eligibility criteria of precision medicine clinical trials via genetic biomarkers and apply it to patients undergoing treatment at the Stanford Cancer Center. This study demonstrates, through our patient eligibility screening algorithm that leverages clinical sequencing derived biomarkers with precision medicine clinical trials, the successful use of an automated algorithmic pipeline as a feasible, accurate and effective alternative to the traditional manual clinical trial curation.
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March 2021

Increased risk of lymphoid malignancy in patients with herpes zoster: a longitudinal follow-up study using a national cohort.

BMC Cancer 2019 Nov 27;19(1):1148. Epub 2019 Nov 27.

Department of Laboratory Medicine, Hallym University College of Medicine, Anyang, Republic of Korea.

Background: The association between herpes zoster and the risk of lymphoid neoplasms in Asian populations has not yet been established. We performed a longitudinal follow-up study using a nationwide cohort to assess the risk of lymphoid neoplasms arising after herpes zoster infection in the adult Korean population.

Methods: Data from participants ≥20 years of age who were registered in the Korean National Health Insurance Service-National Sample Cohort database between 2002 and 2013 were collected. We extracted the data of participants with herpes zoster (n = 59,495) as well as those of matched references at a ratio of 1:4 (n = 237,980) and investigated the subsequent occurrence of lymphoid neoplasms. A stratified Cox proportional hazards model was used to calculate unadjusted hazard ratios (HRs) as well as those adjusted for the Charlson comorbidity index score.

Results: The rate of lymphoid neoplasms was higher in the herpes zoster group (0.15% [90/59,495]) than in the reference group (0.08% [212/237,980], P < 0.001). The unadjusted and adjusted HRs of herpes zoster in patients with lymphoid neoplasms were 1.68 (95% confidence interval [CI] = 1.31-2.15) and 1.58 (95% CI = 1.23-2.02), respectively (P < 0.001 for both). On subgroup analyses according to age and sex, herpes zoster was associated with an increased risk of lymphoid neoplasms in all subgroups; the adjusted HRs were 1.53 (95% CI = 1.05-2.24) for patients < 60 years old, 1.58 (95% CI = 1.14-2.20) for patients ≥60 years old, 1.64 (95% CI = 1.16-2.31) for men, and 1.51 (95% CI = 1.06-2.16) for women (P < 0.05 for all). On subgroup analysis of lymphoid neoplasm subtypes, herpes zoster was associated with the risk of Hodgkin's disease (adjusted HR: 3.23 [95% CI = 1.17-8.93]) and multiple myeloma/malignant plasma cell neoplasms (adjusted HR: 2.17 [95% CI = 1.33-3.54]) (P < 0.05 for both).

Conclusion: Herpes zoster is associated with lymphoid neoplasm development in the Korean population irrespective of age and sex. The risks of Hodgkin's disease and plasma cell neoplasms are significantly elevated in patients with herpes zoster.
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http://dx.doi.org/10.1186/s12885-019-6349-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882027PMC
November 2019

Targeted deep sequencing of cell-free DNA in serous body cavity fluids with malignant, suspicious, and benign cytology.

Cancer Cytopathol 2020 01 21;128(1):43-56. Epub 2019 Nov 21.

Department of Pathology, Stanford University School of Medicine, Stanford, California.

Background: Liquid biopsy using cell-free DNA (cfDNA) presents new opportunities for solid tumor genotyping. While studies have demonstrated the utility of cfDNA from plasma, cfDNA from other body fluids remains underexplored.

Methods: We evaluated the molecular features and clinicopathologic correlates of cfDNA from serous body cavity fluids by performing hybrid capture-based next-generation sequencing (NGS) on cfDNA isolated from residual effusion supernatants. Twenty-one serous effusions from pleural (n = 15), peritoneal (n = 5), and pericardial (n = 1) cavity were analyzed.

Results: The supernatants provided a median cfDNA concentration of 10.3 ng/µL. Notably, all effusions were sequenced successfully to a median depth >1000×, revealing a broad range of genetic alterations including single nucleotide variants, small insertions and deletions, amplifications, and fusions. Specifically, pathogenic alterations were identified in all malignant fluids (13/13), all fluids suspicious for malignancy (2/2), and 1 benign fluid (1/6) from a patient with metastatic cancer. To validate our findings, we examined matching results from 11 patients who underwent additional testing using formalin-fixed, paraffin-embedded (FFPE) specimens. In 8 patients, the paired results between FFPE and supernatant testing were concordant, whereas in the remaining 3 patients, supernatant analysis identified additional variants likely associated with resistance to targeted therapies. Additional comparison between FFPE and supernatant testing showed no difference in DNA concentration (P = .5), depth of coverage (P = .6), or allele frequency of pathogenic mutations (P = .7).

Conclusion: cfDNA isolated from serous body cavity fluids represents a promising source of genomic input for targeted NGS.
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http://dx.doi.org/10.1002/cncy.22205DOI Listing
January 2020

Antiphospholipid Antibodies in Patients With Lupus Anticoagulant Prozone Effect.

Am J Clin Pathol 2020 01;153(2):229-234

Department of Pathology, School of Medicine, Stanford University, Stanford, CA.

Objectives: Lupus anticoagulant (LAC) is typically associated with thrombosis but also rarely with hemorrhage. Some patients exhibit a prozone effect on LAC testing. Antiphosphatidylserine/prothrombin (aPS/PT) antibodies may provide a mechanism for both hemorrhage and prozone effect. Our goal was to evaluate whether antibody specificities, isotypes, and titers were associated with LAC prozone effect, factor II levels, hemorrhage, and thrombosis.

Methods: Patients with prozone effect noted on LAC testing were entered into a database over 3 years. Factor II activity and aPS/PT antibody testing were performed when a sufficient residual sample was available.

Results: All patients with LAC prozone effect and antibody testing were positive for at least 1 class of aPS/PT antibodies. In addition, aPS/PT IgG titers were significantly associated with thrombosis and significantly inversely associated with factor II levels.

Conclusions: In prozone effect patients, aPS/PT antibodies are associated with LAC prozone effect as well as thrombosis and decreased factor II levels.
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http://dx.doi.org/10.1093/ajcp/aqz151DOI Listing
January 2020

High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.

Blood Adv 2019 10;3(19):2825-2835

Department of Pathology.

Chromosomal rearrangements involving the mixed lineage leukemia () gene, also known as , are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that -rearranged () mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
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http://dx.doi.org/10.1182/bloodadvances.2019000450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784514PMC
October 2019

Molecular profiling of clear cell adenocarcinoma of the urinary tract.

Virchows Arch 2019 Dec 2;475(6):727-734. Epub 2019 Aug 2.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Clear cell adenocarcinoma (CCA) of the urinary tract is a rare type of malignancy whose molecular profiles remain undefined. Here we reported an integrated clinicopathologic and molecular profiling analysis of four cases of clear cell adenocarcinoma arising in the urethra or the bladder. Utilizing a clinically validated 130-gene exon-sequencing assay, we identified recurrent pathogenic PIK3CA (p. E545K) and KRAS (p.G12D) variants in three of four (75%) of the cases. In addition, an APC variant (P.S2310X), a TP53 variant (p.R273C), and a MYC amplification event were identified. The only CCA case without either PIK3CA or KRAS variants has a distinct pathogenesis through BK virus, demonstrated by positive BK virus PCR and SV40 immunohistochemistry. The novel finding of recurrent variants in the PI3K/AKT/mTOR pathway provides not only insights into oncogenesis but also potential clinical therapeutic targets for patients with clear cell adenocarcinoma of the urinary tract.
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http://dx.doi.org/10.1007/s00428-019-02634-5DOI Listing
December 2019

Indolent In Situ B-Cell Neoplasms With MYC Rearrangements Show Somatic Mutations in MYC and TNFRSF14 by Next-generation Sequencing.

Am J Surg Pathol 2019 12;43(12):1720-1725

Departments of Pathology.

Systemic high-grade B-cell lymphomas (HGBCLs) with MYC gene rearrangements are clinically aggressive. In situ lesions with indolent behavior have not been described to date. We have identified 2 cases of in situ B-cell neoplasms with MYC rearrangements (IS-BCN, MYC) occurring, and focally confined to ≤4 lymphoid follicles in otherwise healthy individuals and without clinical progression despite minimal intervention (surgical only). Morphologically similar to systemic HGBCLs, the low power view of these lesions showed a starry sky pattern with numerous mitotic figures. High power imaging demonstrated these cells to be medium-large in size with irregular nuclear contours, immature chromatin, and prominent nucleoli. Immunophenotypically these cells were light chain restricted, positive for CD20, CD10, c-Myc, and dim or negative for BCL2 with a Ki67 proliferative index of >95%. By fluorescence in situ hybridization studies, we detected MYC translocations in these cells but no rearrangements in BCL2 or BCL6. Microdissection of neoplastic cells in these patients followed by targeted next-generation sequencing identified a mutation in MYC, D2N, and an indel in TNFRSF14. Mutations in ID3 or TCF3 were not identified. Although rare, these lesions should be separated from HGBCLs involving follicles but with systemic spread which has been previously described. Unlike systemic lymphomas with MYC gene rearrangements, these in situ B-cell neoplasms with MYC rearrangements did not require systemic therapy and no progression has been seen in either patient beyond 1 year (29 and 16 mo). Our work offers pathologic and biologic insight into the early process of B-cell neoplasia.
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http://dx.doi.org/10.1097/PAS.0000000000001338DOI Listing
December 2019

Implementation of Whole-Blood Impedance Aggregometry for Heparin-Induced Thrombocytopenia Functional Assay and Case Discussion.

Am J Clin Pathol 2019 06;152(1):50-58

Department of Pathology, Stanford University, Stanford, CA.

Objectives: The diagnosis of heparin-induced thrombocytopenia (HIT) ideally requires a functional assay to confirm. 14C-serotonin release assay (SRA) as "gold standard" is technically challenging and unsuitable for routine use. We conducted a study to assess the performance of whole-blood impedance aggregometry (WBIA) as a simple and rapid HIT functional assay.

Methods: Platelet factor 4 (PF4)/immunoglobulin G (IgG) antibody, WBIA, and SRA were tested on 70 patients suspected of having HIT. Patients with a 4Ts score of 4 or more, positive PF4/IgG, and positive SRA were considered HIT positive; others were designated HIT negative.

Results: WBIA had 85.7% (6/7) sensitivity and 98.4% (61/62) specificity, which were not statistically different compared with SRA. Sixty-two of 70 patients had concordant results (five positive and 57 negative) by both WBIA and SRA. Eight discordant cases revealed the importance of recognizing donor effect, interferences, and the presence of heparin-independent or non-heparin-dependent antibodies in functional assays.

Conclusions: Implementation of WBIA could facilitate timely diagnosis and management of HIT.
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http://dx.doi.org/10.1093/ajcp/aqz013DOI Listing
June 2019

Update on diagnostic testing for platelet function disorders: What is practical and useful?

Int J Lab Hematol 2019 May;41 Suppl 1:26-32

Departments of Pathology and Medicine, Stanford University, Stanford, California.

Introduction: Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis.

Methods: This review summarizes the authors' experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD.

Results: Blood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high-quality evidence, limit their ability to offer an expanded test menu for PFD.

Conclusion: PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.
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http://dx.doi.org/10.1111/ijlh.12995DOI Listing
May 2019

Revisiting diagnostic criteria for myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis: Borderline cases without anemia exist.

Int J Lab Hematol 2019 Jun 27;41(3):345-352. Epub 2019 Feb 27.

Department of Pathology, Stanford University Medical Center, Stanford, California.

Introduction: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare disease in the 2016 revised World Health Organization (WHO) classification. Diagnostic criteria include the following: persistent thrombocytosis (>450 × 10 /L) with clustering of atypical megakaryocytes, refractory anemia, dyserythropoiesis with ring sideroblasts, and the presence of the spliceosome factor 3b subunit (SF3B1) mutation. It is unclear if anemia should be a required criterion for this diagnosis as cases which show all other features of MDS/MPN-RS-T but without anemia exist.

Methods: We searched for borderline cases of MDS/MPN-RS-T in which refractory anemia was absent at diagnosis in two major academic institutes.

Results: Three cases without anemia were identified. These cases all showed other classic morphologic and clinical features of MDS/MPN-RS-T, including thrombocytosis, atypical megakaryocytes with clustering, and characteristic SF3B1 and JAK2 V617F mutations.

Conclusion: Given these findings, the requirement of refractory anemia as a diagnostic criterion for MDS/MPN-RS-T should be re-evaluated. Removal of refractory anemia as a diagnostic criterion would incorporate current borderline cases and extend the spectrum of this disorder.
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http://dx.doi.org/10.1111/ijlh.12981DOI Listing
June 2019

Structural Variation Detection by Proximity Ligation from Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

J Mol Diagn 2019 05 31;21(3):375-383. Epub 2018 Dec 31.

Department of Pathology, Stanford University School of Medicine, Stanford, California; Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, California. Electronic address:

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.
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http://dx.doi.org/10.1016/j.jmoldx.2018.11.003DOI Listing
May 2019

leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing.

Blood Adv 2018 04;2(8):832-845

Department of Pathology, Stanford University, Stanford, CA.

Genome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here, we used transcription activator-like effector nucleases to generate t(9;11) chromosomal translocations encoding and reciprocal fusion products in CD34 human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated target gene expression, high surface CD9 expression, and increased colony-forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient -rearranged leukemias. No secondary pathogenic mutations were revealed by targeted exome sequencing and whole genome RNA-sequencing analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute rearranged leukemia in vivo, reflecting the genetic simplicity of this disease, and provides an experimental platform for biological and disease-modeling applications.
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http://dx.doi.org/10.1182/bloodadvances.2017013748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916000PMC
April 2018

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

Int J Gynecol Pathol 2019 Jul;38(4):386-392

Department of Pathology (M.T., D.F.S., J.L.Z., A.K.F.) Department of Obstetrics and Gynecology (A.K.K.) Department of Medicine (J.L.Z.), Stanford University School of Medicine, Stanford Department of Obstetrics and Gynecology, UCSF Fresno, Fresno (M.T.), California.

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.
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http://dx.doi.org/10.1097/PGP.0000000000000507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170737PMC
July 2019

Next-generation sequencing of idiopathic multicentric and unicentric Castleman disease and follicular dendritic cell sarcomas.

Blood Adv 2018 03;2(5):481-491

Department of Pathology, Stanford University, Stanford, CA.

Castleman disease (CD) is a rare lymphoproliferative disorder subclassified as unicentric CD (UCD) or multicentric CD (MCD) based on clinical features and the distribution of enlarged lymph nodes with characteristic histopathology. MCD can be further subtyped based on human herpes virus 8 (HHV8) infection into HHV8-associated MCD, HHV8/idiopathic MCD (iMCD), and polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin change (POEMS)-associated MCD. In a subset of cases of UCD, an associated follicular dendritic cell sarcoma (FDCS) may be seen. Although numerous reports of the clinical and histologic features of UCD, MCD, and FDCS exist, an understanding of the genetic and epigenetic landscape of these rare diseases is lacking. Given this paucity of knowledge, we analyzed 15 cases of UCD and 3 cases of iMCD by targeted next-generation sequencing (NGS; 405 genes) and 3 cases of FDCS associated with UCD hyaline vascular variant (UCD-HVV) by whole-exome sequencing. Common amplifications of , , and were seen in 1 iMCD and 1 UCD case; the iMCD case also had a somatic L295Q mutation. This iMCD patient also showed clinicopathologic features consistent with a specific subtype known as Castleman-Kojima disease (thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly [TAFRO] clinical subtype). Additionally, 1 case of UCD-HVV showed amplification of the cluster of histone genes on chromosome 6p. FDCS associated with UCD-HVV showed mutations and copy number changes in known oncogenes, tumor suppressors, and chromatin structural-remodeling proteins.
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http://dx.doi.org/10.1182/bloodadvances.2017009654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851414PMC
March 2018

Comprehensive Genomic Profiling of Malignant Effusions in Patients with Metastatic Lung Adenocarcinoma.

J Mol Diagn 2018 03 19;20(2):184-194. Epub 2017 Dec 19.

Department of Pathology, Stanford University School of Medicine, Stanford, California. Electronic address:

Cytology samples are increasingly used for comprehensive molecular testing. Although fine-needle aspirates are adequate substrates for high-throughput sequencing, the suitability of malignant body fluids remains largely unexplored. We investigated the adequacy and utility of performing targeted next-generation sequencing (NGS) on malignant effusions from patients with metastatic lung adenocarcinoma. Thirty-two effusion samples submitted for hybrid capture-based NGS using a clinically validated solid tumor genotyping panel were examined. All cases showed ≥5% tumor cellularity; however, 28 (88%) provided sufficient DNA for NGS (≥1 ng/μL). The sequencing reads showed satisfactory quality control statistics, and the variant allele frequencies were correlated with tumor cellularity. Furthermore, pathogenic or likely pathogenic genomic alterations were identified in 26 of 28 samples (93%), whereas clinically actionable alterations were present in 18 (64%). Notably, nine patients had additional molecular testing performed on preceding or subsequent biopsy specimens, and the results across multiple samples were compared. In two patients, the NGS-based fluid analysis identified clinically actionable alterations that were not detected by other hotspot testing. In four patients treated with tyrosine kinase inhibitors, malignant fluid sequencing confirmed driver alterations from prior testing and revealed new resistance mechanisms. Hence, given adequate DNA input and tumor cellularity, comprehensive genomic profiling of malignant effusions may be used to establish mutational status at diagnosis and inform treatment resistance during targeted therapy.
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http://dx.doi.org/10.1016/j.jmoldx.2017.10.007DOI Listing
March 2018
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