Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.

BACKGROUND: Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vß repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant ß chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. METHODS: Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. RESULTS: We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. CONCLUSIONS: Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells.

BACKGROUND: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. METHODS: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. RESULTS: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. CONCLUSION: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. TRIAL REGISTRATION: NCT03384108 and NCT03119883.

Rapid assessment of hyperdiploidy in plasma cell disorders using a novel multi-parametric flow cytometry method.

Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19-1.50) versus those with low-hyperdiploidy (DNA index: 1.05-1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.

Flow Cytometric Assessment of Chronic Myeloid Neoplasms.

Flow cytometry immunophenotyping of the hematopoietic cells from the bone marrow can help with diagnosis, prognosis, and therapy of chronic myeloid neoplasms. Unlike with B-cell neoplasms, there is no simple phenotypic test to substitute for clonality. Therefore, antigen panels to evaluate myeloid neoplasms are larger, and the gating strategies more complex than for lymphoid neoplasms. The number of phenotypic abnormalities in hematopoietic cells correlates with disease severity and cytogenetic complexity, and can be integrated into a scoring system for diagnostic and prognostic purposes. However, flow cytometry remains only an adjunct diagnostic modality.