15-color highly sensitive flow cytometry assay for post anti-CD19 targeted therapy (anti-CD19-CAR-T and blinatumomab) measurable residual disease assessment in B-lymphoblastic leukemia/lymphoma: Real-world applicability and challenges.

OBJECTIVES: Measurable residual disease (MRD) is the most relevant predictor of disease-free survival in B-cell acute lymphoblastic leukemia (B-ALL). We aimed to establish a highly sensitive flow cytometry (MFC)-based B-ALL-MRD (BMRD) assay for patients receiving anti-CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low-level mimics and confounding populations. METHODS: We standardized a 15-color highly-sensitive BMRD assay with an alternate CD19-free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B-ALL patients considered for anti-CD19 immunotherapy. RESULTS: The 15-color BMRD assay with CD22/CD24/CD81/CD33-based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low-level cells that showed immunophenotypic overlap with leukemic B-blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19-/CD10-/CD24- early lymphoid progenitor/precursor type-1 cells (ELP-1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19-/CD24- stage-0 B-cell precursors or ELP-2 cells. CONCLUSIONS: We standardized a highly sensitive 15-color BMRD assay with a non-CD19-based gating strategy for patients receiving anti-CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low-level populations that can be misinterpreted as MRD in real-world practice.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

Comprehensive immune cell profiling depicts an early immune response associated with severe coronavirus disease 2019 in cancer patients.

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection-associated respiratory disease [coronavirus disease 2019 (COVID-19)]. Some of them were associated with immunopathogenesis of severe COVID-19. However, reports on immunological indicators of severe COVID-19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune-cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID-19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID-19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID-19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T-cell subsets such as regulatory T cells (Tregs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID-19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, Tregs , Th9, effector NK cells, B cells, intermediate-type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID-19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID-19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID-19 in cancer patients without intensive chemo/immunotherapy.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis.

Clusterin (CLU), a multifunctional chaperonic glycoprotein associated with diverse cellular functions has been shown to act as an oncogene or tumour suppressor gene in different cancers, implying a dual role in tumorigenesis. Here, we investigated the expression of CLU isoforms, their subcellular localization and functional significance in oral cancer cells. Significant downregulation of secretory CLU (sCLU) transcripts was observed in oral cancer cell lines and tumours versus normal cells while the nuclear CLU (nCLU) transcripts were undetectable. We demonstrated for the first time the nucleolar localization of sCLU, its response to different nucleolar stresses and association with cajal bodies post nucleolar stress. Functionally, knockdown of CLU revealed its negative association with ribosome biogenesis implying a possible tumour suppressor like role in oral cancers. Further, loss of sCLU in these cells also resulted in altered nuclear morphology and shrunken tubulin filaments. In addition, the levels of nucleolar Nucleophosmin 1(NPM1) and Fibrillarin, known to regulate nuclear morphology were downregulated indicating a possible role of sCLU in their stabilization. Further, an in silico docking approach to gain insights into the interaction of sCLU with nucleolar proteins NPM1, Fibrillarin, UBF and Nucleolin, revealed the involvement of a conserved region comprising of amino acid residues 140-155 of sCLU ß-chain, specifically via the Phe152 residue in hydrophobic interactions with these client nucleolar proteins indicating a possible stabilizing or regulatory role of sCLU. SIGNIFICANCE OF THE STUDY: This is the first study to demonstrate the nucleolar localization of sCLU and its associated functions in oral cancer cells. Downregulation of sCLU in oral cancer tissues and cell lines, and its negative association with ribogenesis suggest its tumour suppressor like role in oral cancers. The possible role of sCLU in stabilization or regulation of different nucleolar proteins thereby impacting their functions is also implicated.