Monitoring malignant T-cell clones by direct TCR expression assay in patients with leukemic cutaneous T-cell lymphoma during extracorporeal photopheresis.

BACKGROUND/PURPOSE: Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vß usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. METHODS: Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n = 15) and during therapy at 3 months and 6 months (n = 12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vß family members. RESULTS: At baseline, DTEA detected TCR-Vß clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vß clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vß clones, DTEA also detected additional TCR-Vß clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVß usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vß and TCRVα usages over therapy were consistent with patients' clinical responses. CONCLUSIONS: Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vß usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

CD209+ monocyte-derived myeloid dendritic cells were increased in patients with leukemic cutaneous T-cell lymphoma undergoing extracorporeal photopheresis via the CELLEXTM system.

BACKGROUND/PURPOSE: We previously reported that myeloid dendritic cells (mDC) were increased in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) following extracorporeal photopheresis (ECP) using the Therakos UVAR XTS™ system. We now assessed monocyte-derived mDCs (Mo-DCs) in L-CTCL patients treated with the CELLEXTM photopheresis system. CD209, a transmembrane receptor, was used to define Mo-DCs. METHODS: Peripheral blood samples from baseline pre-ECP and at Day 2, 1 month, 3 months, and 6 months post-ECP were analyzed by flow cytometry for Lin- HLA-DR+ CD123+ plasmacytoid dendritic cells (pDCs), Lin- HLA-DR+ CD11c+ mDCs, and CD209+ mDCs. The expression of CD209 mRNA was assessed by real-time PCR. RESULTS: At baseline, 7 of 19 patients had lower than normal mDCs, and all patients had lower than normal CD209+ mDCs in peripheral blood mononuclear cells (0.005% in patients, n = 19, vs 0.50% in healthy donors, n = 7, P < .0001). The CD209+ mDC numbers only accounted for 3.28% out of total mDCs in patients compared with 66.51% in healthy donors. After treatment, the CD209+ mDC numbers showed increasing trends in patients. The average absolute numbers of CD209+ mDCs went up by 4.8-fold at 3 months (n = 10, P = .103) and by 6.4-fold at 6 months (n = 9, P = .100). CD209 mRNA expression went up in two patients responsive to therapy, parallel to CD209+ mDC numbers. L-CTCL patients achieved 70% overall clinical response rate (7/10) following ECP therapy with the CELLEXTM system. CONCLUSIONS: Our results suggest that the CELLEXTM photopheresis system is effective for treating L-CTCL patients like the UVAR XTS™ system, and in vivo-generated Mo-DCs increase following ECP.

Small molecule CCR4 antagonists in cutaneous T-cell lymphoma.

Mycosis fungoides (MF) and Sézary syndrome (SS) are two most common types of cutaneous T-cell lymphoma (CTCL). Despite advances in understanding the pathogenesis of MF and SS, effective treatments remain limited. CC chemokine receptor 4 (CCR4) is highly expressed on CTCL cells and serves as a great therapeutic target. Mogamulizumab, an FDA-approved anti-CCR4 antibody, has shown efficacy in treating MF/SS; however, its side effects have raised concerns, underscoring the need for more effective and less toxic CCR4-targeted therapies. While small molecule CCR4 antagonists have been studied in other diseases involving CCR4+ Th2 cells and regulatory T-cells, but their effects in CTCL have not been previously explored. This study assessed the effects of two small molecule CCR4 antagonists, C021 (Class I) and AZD2098 (Class II), in MF derived cell line (MJ) and SS derived cell line (HuT 78) in vitro and in vivo. As results, both C021 and AZD2098 inhibited chemotactic responses to CCL17 and CCL22 in MJ and HuT 78 cells. However, only C021 downregulated CCR4 expression, inhibited cell proliferation, induced cell apoptosis and cell cycle arrest, and decreased colony formation in MJ and HuT 78 cells in vitro. Furthermore, only C021 inhibited tumor growth in CTCL xenograft mice in vivo. These findings suggest that Class I CCR4 antagonists such as C021 exerts more potent anti-tumor effects on CTCL cells in vitro and in vivo compared to Class II CCR4 antagonists like AZD2098, highlighting its potential for clinical application.

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

Extracorporeal photopheresis (ECP) is a frontline therapy for patients with leukemic cutaneous T-cell lymphoma (L-CTCL), but its mechanisms of action are not fully understood. This study was to explore the molecular mechanisms underlying clinical response versus non-response in patients with L-CTCL. We performed blood transcriptional profiling of ten L-CTCL patients at Day 2 and 1 month post- ECP compared to pre-ECP baseline using Agilent Whole Human Genome Microarray technology. Differentially expressed genes (DEGs) between five clinically-responsive patients and five clinically-resistant patients were cross-compared. Higher numbers of genes were modulated in responders than non-responders after ECP at both Day 2 and 1 month, with two thirds of DEGs down-regulated. The down-regulated DEGs at 1 month post-ECP were related to inflammatory, immune and/or stress responses, platelet functions, and chromatin remodeling. Upregulated DEGs were mainly related to functions of the nucleolus. Pathway analysis revealed that integrin and IL-1 signaling pathways were the top pathways affected in responders, which were minimally affected in non-responders. The top upstream transcription regulators affected were IL1B, EGR1, FAS, and TGFB1. Our results suggest that the modulation of cell adhesion and suppression of IL-1ß induced inflammation may underlie the efficacy of ECP in L-CTCL.

Correction: Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

[This corrects the article DOI: 10.18632/oncotarget.26900.].