Benign clonal T-cell expansions in reactive immune responses often complicate the laboratory diagnosis T-cell neoplasia. We recently introduced a novel flow cytometry assay to detect T-cell clones in ...blood and bone marrow, based on the identification of a monophasic T-cell receptor (TCR) β chain constant region-1 (TRBC1) expression pattern within a phenotypically distinct TCRαβ T-cell subset. In routine laboratory practice, T-cell clones of uncertain significance (T-CUS) were detected in 42 of 159 (26%) patients without T-cell malignancy, and in 3 of 24 (13%) healthy donors. Their phenotype (CD8
/CD4
: 78%, CD4
/CD8
: 12%, CD4
/CD8
: 9%, or CD4
/CD8
: 2%) closely resembled that of 26 cases of T-cell large granular lymphocytic leukemia (T-LGLL) studied similarly, except for a much smaller clone size (p < 0.0001), slightly brighter CD2 and CD7, and slightly dimmer CD3 expression (p < 0.05). T-CUS was not associated with age, gender, comorbidities, or peripheral blood counts. TCR-Vβ repertoire analysis confirmed the clonality of T-CUS, and identified additional clonotypic CD8-positive subsets when combined with TRBC1 analysis. We hereby report the phenotypic features and incidence of clonal T-cell subsets in patients with no demonstrable T-cell neoplasia, providing a framework for the differential interpretation of T-cell clones based on their size and phenotypic properties.
Fluorescence in situ hybridization (FISH) is currently the gold-standard assay to detect recurrent genomic abnormalities of prognostic significance in multiple myeloma (MM). Since most translocations ...in MM involve a position effect with heterogeneous breakpoints, we hypothesize that FISH has the potential to miss translocations involving these regions. We evaluated 70 bone marrow samples from patients with plasma cell dyscrasia by FISH and whole-genome mate-pair sequencing (MPseq). Thirty cases (42.9%) displayed at least one instance of discordance between FISH and MPseq for each primary and secondary abnormality evaluated. Nine cases had abnormalities detected by FISH that went undetected by MPseq including 6 tetraploid clones and three cases with missed copy number abnormalities. In contrast, 19 cases had abnormalities detected by MPseq that went undetected by FISH. Seventeen were MYC rearrangements and two were 17p deletions. MPseq identified 36 MYC abnormalities and 17 (50.0% of MYC abnormal group with FISH results) displayed a false negative FISH result. MPseq identified 10 cases (14.3%) with IgL rearrangements, a recent marker of poor outcome, and 10% with abnormalities in genes associated with lenalidomide response or resistance. In summary, MPseq was superior in the characterization of rearrangement complexity and identification of secondary abnormalities demonstrating increased clinical value compared to FISH.
Flow cytometric (FC) immunophenotyping is critical but time-consuming in diagnosing minimal residual disease (MRD). We evaluated whether human-in-the-loop artificial intelligence (AI) could improve ...the efficiency of clinical laboratories in detecting MRD in chronic lymphocytic leukemia (CLL). We developed deep neural networks (DNN) that were trained on a 10-color CLL MRD panel from treated CLL patients, including DNN trained on the full cohort of 202 patients (F-DNN) and DNN trained on 138 patients with low-event cases (MRD < 1000 events) (L-DNN). A hybrid DNN approach was utilized, with F-DNN and L-DNN applied sequentially to cases. “Ground truth” classification of CLL MRD was confirmed by expert analysis. The hybrid DNN approach demonstrated an overall accuracy of 97.1% (95% CI: 84.7−99.9%) in an independent cohort of 34 unknown samples. When CLL cells were reported as a percentage of total white blood cells, there was excellent correlation between the DNN and expert analysis r > 0.999; Passing−Bablok slope = 0.997 (95% CI: 0.988−0.999) and intercept = 0.001 (95% CI: 0.000−0.001). Gating time was dramatically reduced to 12 s/case by DNN from 15 min/case by the manual process. The proposed DNN demonstrated high accuracy in CLL MRD detection and significantly improved workflow efficiency. Additional clinical validation is needed before it can be fully integrated into the existing clinical laboratory practice.
Patients with large granular lymphocytic leukemia (LGLL) frequently present with neutropenia. When present, anemia is usually accompanied by neutropenia and/or thrombocytopenia and isolated anemia is ...uncommon. We evaluated a cohort of 244 LGLL patients spanning 15 years and herein report the clinicopathologic features of 34 (14%) with isolated anemia. The patients with isolated anemia showed a significantly male predominance (p = 0.001), a lower level of hemoglobulin (p < 0.0001) and higher MCV (p = 0.017) and were less likely to have rheumatoid arthritis (p = 0.023) compared to the remaining 210 patients. Of the 34 LGLL patients with isolated anemia, 13 (38%) presented with pure red cell aplasia (PRCA), markedly decreased reticulocyte count and erythroid precursors, and more transfusion-dependence when compared to non-PRCA patients. There was no other significant clinicopathologic difference between PRCA and non-PRCA patients. 32 patients were followed for a median duration of 51 months (6-199). 24 patients were treated (11/11 PRCA and 13/21 non-PRCA patients, p < 0.02). The overall response rate to first-line therapy was 83% 8/11 (72.7%) for PRCA, 12/13 (92.3%) for non-PRCA, including 14 showing complete response and 6 showing partial response with a median response duration of 48 months (12-129). Half of non-PRCA patients who were observed experienced progressive anemia. During follow-up, no patients developed neutropenia; however, 5/27 (18.5%) patients developed thrombocytopenia. No significant difference in overall survival was noted between PRCA and non-PRCA patients. In summary, this study demonstrates the unique features of LGLL with isolated anemia and underscores the importance of recognizing LGLL as a potential cause of isolated anemia, which may benefit from disease-specific treatment. LGLL patients with PRCA were more likely to require treatment but demonstrated similar clinicopathologic features, therapeutic responses, and overall survival compared to isolated anemia without PRCA, suggesting PRCA and non-PRCA of T-LGLL belong to a common disease spectrum.
As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute ...to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1–signaling pathway that stabilizes interleukin-2 (IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome–derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4–signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.
•T-cell activation induces TCR transactivation of CXCR4 to stabilize cytokine mRNA transcripts via a PREX1-Rac1–signaling pathway.•Inhibition of the TCR-CXCR4–signaling pathway impairs TCR-dependent and TCR-independent cytokine secretion by CTCL cells.
Circulating plasma cells at diagnosis, prior to auto-transplant and at relapse have a negative impact on survival in multiple myeloma. However, the impact of kinetics of circulating plasma cells ...along the course of illness has not been defined. We have analyzed 247 newly diagnosed multiple myeloma patients undergoing early auto-transplant who had paired evaluation of circulating plasma cells at diagnosis and pre-transplant by 6-color flow cytometry. A total of 117 patients had no detectable circulating plasma cells at both time points (CPC-/-), 82 had circulating plasma cells at diagnosis followed by complete eradication after induction (CPC+/-) and 48 had circulating plasma cells at transplant, including persistence of cells (CPC+/+; n=45) or emergence of new cells (CPC-/+; n=3) after induction. The rate of post-transplant stringent complete response was 32% in the CPC-/-, 30% in CPC+/- and 12% in CPC+/+ or -/+ groups (
=0.018). At a median follow up of 58 months from transplantation, the median progression-free survival in the 3 respective groups were 30, 24 and 14 months, and the 5-year overall survival rates were 83%, 70% and 43% (
<0.001 for both comparisons). On a multivariate analysis for overall survival, the risk of mortality was higher in CPC +/- (hazard ratio 2.7, 95%CI: 1.3-5.8;
=0.009) and CPC+/+ or -/+ (hazard ratio 5.7, 95%CI: 2.5-13.1;
<0.001) groups compared to the CPC-/- group. Monitoring for circulating plasma cells before induction therapy and before transplant by 6-color flow cytometry is predictive of survival in newly diagnosed myeloma and should be incorporated into clinical trials.
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