STAT3 mutations are frequent in CD30+ T-cell lymphomas and T-cell large granular lymphocytic leukemia.
2013; 27 (11): 2244-2247
Feasibility of using microbeads with holographic barcodes to track DNA specimens in the clinical molecular laboratory.
Next-generation sequencing in hematologic malignancies: what will be the dividends?
Therapeutic advances in hematology
2012; 3 (6): 333-339
We demonstrate the feasibility of using glass microbeads with a holographic barcode identifier to track DNA specimens in the molecular pathology laboratory. These beads can be added to peripheral blood specimens and are carried through automated DNA extraction protocols that use magnetic glass particles. We found that an adequate number of microbeads are consistently carried over during genomic DNA extraction to allow specimen identification, that the beads do not interfere with the performance of several different molecular assays, and that the beads and genomic DNA remain stable when stored together under regular storage conditions in the molecular pathology laboratory. The beads function as an internal, easily readable specimen barcode. This approach may be useful for identifying DNA specimens and reducing errors associated with molecular laboratory testing.
View details for DOI 10.7717/peerj.91
View details for PubMedID 23862106
Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms
2010; 116 (6): 988-992
The application of high-throughput, massively parallel sequencing technologies to hematologic malignancies over the past several years has provided novel insights into disease initiation, progression, and response to therapy. Here, we describe how these new DNA sequencing technologies have been applied to hematolymphoid malignancies. With further improvements in the sequencing and analysis methods as well as integration of the resulting data with clinical information, we expect these technologies will facilitate more precise and tailored treatment for patients with hematologic neoplasms.
View details for DOI 10.1177/2040620712458948
View details for PubMedID 23606936
Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements
JOURNAL OF IMMUNOLOGY
2010; 184 (12): 6986-6992
Dysregulated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F-negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34(+) early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.
View details for DOI 10.1182/blood-2010-02-270108
View details for Web of Science ID 000280881700021
View details for PubMedID 20404132
Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory
JOURNAL OF MOLECULAR DIAGNOSTICS
2010; 12 (1): 58-64
Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.
View details for DOI 10.4049/jimmunol.1000445
View details for Web of Science ID 000278516700047
View details for PubMedID 20495067
Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing
SCIENCE TRANSLATIONAL MEDICINE
2009; 1 (12)
The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.
View details for DOI 10.2353/jmoldx.2010.090068
View details for Web of Science ID 000273664100009
View details for PubMedID 19959796
Growth of Histoplasma capsulatum isolates is better on potato dextrose agar with chloramphenicol than on brain heart infusion agar
JOURNAL DE MYCOLOGIE MEDICALE
2009; 19 (3): 197-199
Clinical characterization of acute myeloid leukemia with myelodysplasia-related changes as defined by the 2008 WHO classification system
2009; 113 (9): 1906-1908
The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.
View details for DOI 10.1126/scitranslmed.3000540
View details for Web of Science ID 000277263200001
View details for PubMedID 20161664
Cold agglutinin syndrome in pediatric liver transplant recipients
2007; 11 (8): 931-936
Although some studies have validated the 2001 World Health Organization (WHO) classification of acute myeloid leukemia (AML), including the importance of multilineage dysplasia, others have suggested that multilineage dysplasia correlates with unfavorable cytogenetics but has no independent impact on prognosis. In 2008, the revised WHO classification has expanded this category into "AML with myelodysplasia-related changes" (AML-MRC). We evaluated the clinical, pathologic, cytogenetic, and molecular features of 100 AML patients using the 2008 WHO criteria. Patients underwent genetic screening for NPM1, FLT3-ITD, FLT3-D835, and CEBPA mutations. Compared with patients with AML, not otherwise specified, patients with AML-MRC were significantly older (P= .014), presented with a lower hemoglobin (P= .044), more frequently expressed CD14 (P= .048), and exhibited a decreased frequency of CEBPA mutations (P= .001). Multivariate analysis indicated that patients with AML-MRC had a significantly worse overall survival, progression-free survival, and complete response compared with AML-not otherwise specified (all P< .001). These data support the clinical, morphologic, and cytogenetic criteria for this 2008 WHO AML category.
View details for DOI 10.1182/blood-2008-10-182782
View details for Web of Science ID 000263723700007
View details for PubMedID 19131546
Molecular diagnostics of non-Hodgkin lymphoma.
Expert opinion on medical diagnostics
2007; 1 (1): 47-63
Anemia is a common finding in post-liver transplant patients. Causes for the anemia include nutritional deficiencies, red cell aplasia as well as immune-mediated hemolysis. One of the immunologic causes of hemolytic anemia is drug-induced hemolysis. Tacrolimus is a common immunosuppressant used in post-liver transplant patients to prevent graft rejection. There have been reports of tacrolimus-associated hemolytic anemia secondary to hemolytic uremic syndrome as well as autoimmune hemolysis. There are also case-reports of severe hemolytic anemia related to cold agglutinin production in post-liver transplant patients. We described in this paper three cases of severe cold agglutinin hemolytic anemia in three pediatric liver transplant patients. Steroid therapy, plasmapheresis and withdrawal of tacrolimus led to resolution of the severe hemolytic process in each case. Whether the immune-mediated hemolysis is related to tacrolimus is not clear and needs to be characterized further.
View details for DOI 10.1111/j.1399-3046.2007.00795.x
View details for Web of Science ID 000250520100016
View details for PubMedID 17976131
Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray
JOURNAL OF CLINICAL MICROBIOLOGY
2007; 45 (7): 2340-2343
Non-Hodgkin lymphomas (NHLs) represent a diverse group of hematopoietic neoplasms. Morphology and immunophenotypic characterization are the cornerstone of the diagnosis and classification of NHLs; however, cytogenetic and molecular studies are being increasingly used to provide further information about classification and prognosis. In addition, these molecular techniques are being used for the detection of minimal residual or recurrent disease after the patient has undergone treatment. This review will discuss the major genetic changes associated with NHLs and describe the methods currently used in the clinical laboratory for cytogenetic and molecular detection of these abnormalities.
View details for DOI 10.1517/17530059.1.1.47
View details for PubMedID 23489268
Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult
CLINICAL INFECTIOUS DISEASES
2006; 43 (8): E71-E76
A pan-virus DNA microarray (Virochip) was used to detect a human metapneumovirus (hMPV) strain associated with a critical respiratory tract infection in an elderly adult with chronic lymphocytic leukemia. This infection had previously eluded diagnosis despite extensive microbiological testing for possible etiologic agents. The patient's hMPV strain did not grow in viral culture, and only one of five specific reverse transcription-PCR assays for hMPV was positive.
View details for DOI 10.1128/JCM.00364-07
View details for Web of Science ID 000248072900050
View details for PubMedID 17494722
A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.
View details for Web of Science ID 000240666200029
View details for PubMedID 16983602