BCR-ABL1 (p210) Transcript Kinetics.

CONTEXT.­: Delta checks are a powerful technique for monitoring clinical assays in many disciplines but have not been routinely used in molecular testing. OBJECTIVE.­: To determine if the biologically determined kinetics of BCR-ABL1's rise and fall could allow the development of a delta check in BCR-ABL1 testing. DESIGN.­: Nine years of BCR-ABL1 p210 results were evaluated, and patients with 3 or more results were selected for inclusion. The kinetics of these percentages of international standard values were plotted against time along with the median and the 90th and 95th percentile lines. A Monte Carlo simulation of a batch mix-up was performed for 6 months of data to determine the efficacy of the proposed cutoff. RESULTS.­: The median kinetics showed a 1-log drop of the percentage of international standard in 90 days, with less than 5% of cases showing faster than a 2-log drop in 90 days, and less than 2.5% showing a faster than 3-log drop in 90 days (extrapolated to 1 log in 30 days). The Monte Carlo simulation of a batch mix-up showed that an average batch mix-up of 23 samples could routinely be flagged by this cutoff, albeit with wide variance. CONCLUSIONS.­: These results suggest that using a drop in the percentage of international standard of greater than 1 log in 30 days can be a useful trigger in implementing a delta-check system for this molecular test.

Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole Blood and Blast-Enriched Samples.

OBJECTIVES: Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. METHODS: Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. RESULTS: Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. CONCLUSIONS: Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.

Current Aspects of Clonal Hematopoiesis: Implications for Clinical Diagnosis.

The broad dissemination of next-generation sequencing capability has increased recognition of clonal hematopoiesis in various clinical settings. In hematologically normal individuals, somatic mutations may occur at an increasing frequency with age in genes that are also commonly mutated in overt myeloid malignancies such as AML and MDS (e.g., DNMT3A, TET2, and ASXL1). This is referred to as clonal hematopoiesis of indeterminate potential (CHIP) and is a benign state; however, it carries a risk of progression to hematologic malignancy as well as mortality primarily because of increased cardiovascular events. In clinical settings, clonal hematopoiesis may be observed in cytopenic patients who do not otherwise meet the criteria for hematologic malignancy, a condition referred to as clonal cytopenias of undetermined significance (CCUS). Distinguishing CCUS from overt MDS or other myeloid neoplasms can be challenging because of the overlapping mutational landscape observed in these conditions. Genetic features that could be diagnostically helpful in making this distinction include the number and biological function of mutated genes as well as the observed variant allele frequency. A working knowledge of clonal hematopoiesis is essential for the diagnosis and clinical management of patients with hematologic conditions. This review describes the key characteristics of clonal hematopoiesis with particular focus on implications for differential diagnosis in patients with CHIP, idiopathic cytopenia, CCUS, and myeloid malignancy.

GATA3 Immunohistochemical Staining in Hodgkin Lymphoma: Diagnostic Utility in Differentiating Classic Hodgkin Lymphoma From Nodular Lymphocyte Predominant Hodgkin Lymphoma and Other Mimicking Entities.

BACKGROUND: Classic Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) are clinically distinct entities, with different prognostic and treatment implications. In addition, several large B-cell lymphomas and some T-cell lymphomas can mimic CHL. Differentiating these entities from CHL is crucial for ensuring appropriate therapy. GATA3 is a T-cell transcription factor involved in T-cell maturation and has been previously shown to be overexpressed in CHL cells via gene expression profiling. We investigated the utility of GATA3 immunostain in differentiating CHL from NLPHL and other mimicking entities. MATERIALS AND METHODS: We accrued 17 NLPHLs, 49 CHLs [23 nodular sclerosis (NS), 3 syncytial variants, 3 lymphocyte rich and 13 mixed cellularity types], 4 primary mediastinal large B-cell lymphomas (PMBLs), 2 Epstein-Barr virus (EBV) positive diffuse large B-cell lymphomas (DLBCLs) (EBV+LBCLs), 2 T-cell/histiocyte-rich large B-cell lymphomas (TCHRBCLs), 1 gray zone lymphoma, and 2 tissue microarrays consisting of 72 DLBCLs. One slide from each was stained with GATA3 and percent positive tumor cells and intensity of nuclear expression was semiquantitatively graded independently by 2 board certified hematopathologists. RESULTS: GATA3 was positive in 80% of CHLs. Both percent positivity and intensity of staining varied greatly. Syncytial variant of NS subtype showed the highest positivity rate (3/3; 100%), followed by NS (20/23; 87%), mixed cellularity (9/13; 70%), and lymphocyte rich (2/3; 67%). GATA3 was negative in all NLPHLs, EBV+LBCLs, TCRBCLs, and DLBCLs stained. The single gray zone lymphoma and 3/4 PMBLs were positive. CONCLUSIONS: Nuclear expression of GATA3 can be used to delineate CHL from NLPHL. GATA3 positivity effectively excludes NLPHL with 100% negative predictive value. However, as 20% of CHL can be negative for GATA3, CHL cannot be ruled out with negative GATA3. Additional findings include GATA3 positivity among PMBLs, whereas all 72 DLBCLs were negative for GATA3. This finding further highlights similarities between CHL and PMBL.

Comparative Analysis Reveals Potential Utility of Digital Microscopy in the Evaluation of Peripheral Blood Smears With Some Barriers to Implementation.

OBJECTIVES: Evaluation of the peripheral blood smear (PBS) is an essential diagnostic test in current medical practice. We aimed to evaluate the use of digital microscopy for the examination of PBS as an option to provide expert interpretation to remote sites and in "on-call" situations. METHODS: We collected 100 Wright-Giemsa-stained PBS slides representing normal and abnormal findings seen at a community-based hospital. Four hematopathologists independently evaluated the cases using conventional light and digital microscopy. RESULTS: When comparing digital vs light microscopy, most of the cellular features evaluated showed at least a moderate degree of agreement in at least three of the reviewers. Discrepancies in final diagnosis were identified in a minority of the cases, most of which were attributed to the poorer resolution of digital microscopy at high magnification (×400). CONCLUSIONS: These results support the limited use of digital microscopy for evaluation and triage of peripheral blood smears as a practical option to obtain expert opinion in locations where experienced staff is not available on site. Our results indicate that while digital microscopy is well suited for basic triage of these blood smears, limitations in quality of imaging at higher magnification as well as large file size may limit its utility in certain settings and situations.