Single-Cell DNA Sequencing Reveals an Evolutionary Pattern of CHIP in Transplant Eligible Multiple Myeloma Patients.

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the phenomenon where a hematopoietic stem cell acquires fitness-increasing mutation(s), resulting in its clonal expansion. CHIP is frequently observed in multiple myeloma (MM) patients, and it is associated with a worse outcome. High-throughput amplicon-based single-cell DNA sequencing was performed on circulating CD34+ cells collected from twelve MM patients before autologous stem cell transplantation (ASCT). Moreover, in four MM patients, longitudinal samples either before or post-ASCT were collected. Single-cell sequencing and data analysis were assessed using the MissionBio Tapestri® platform, with a targeted panel of 20 leukemia-associated genes. We detected CHIP pathogenic mutations in 6/12 patients (50%) at the time of transplant. The most frequently mutated genes were TET2, EZH2, KIT, DNMT3A, and ASXL1. In two patients, we observed co-occurring mutations involving an epigenetic modifier (i.e., DNMT3A) and/or a gene involved in splicing machinery (i.e., SF3B1) and/or a tyrosine kinase receptor (i.e., KIT) in the same clone. Longitudinal analysis of paired samples revealed a positive selection of mutant high-fitness clones over time, regardless of their affinity with a major or minor sub-clone. Copy number analysis of the panel of all genes did not show any numerical alterations present in stem cell compartment. Moreover, we observed a tendency of CHIP-positive patients to achieve a suboptimal response to therapy compared to those without. A sub-clone dynamic of high-fitness mutations over time was confirmed.

Case report: Senescence as mechanism of resistance to Pembrolizumab in a Lymphoma patient who failed CD19-Targeted CAR-T cell therapy.

Background: T cells engineered to target CD19 antigen on neoplastic B cells represent the most striking example of CAR-T cell therapy. The success rate of this therapy is affected by several limitations: target antigen loss, and/or acquisition of a senescent/exhausted phenotype by CAR and non-CAR T cells. Case presentation: We report on a patient affected by refractory Diffuse Large B-cell Lymphoma who was resistant to CAR T-cell therapy and to two cycles post CAR-T of pembrolizumab (PBZ) due to the evolution into a B-cell Hodgkin-like lymphoma. Owing to the CD30 expression and the Hodgkin-like phenotype, the patient was ultimately treated with Brentuximab-Vedotin and finally underwent remission. Upon PBZ treatment, 100% of circulating CAR-T+ cells showed a persistent CD8+ senescent/exhausted phenotype, while an increase in the percentage of senescent cells was found in the non-CAR CD8+ T cells compartment. Conclusions: PBZ is not able to reinvigorate exhausted CAR+ T cells and to confer durable clinical response. We hypothesize that the phenomenon is due to the senescent phenotype of CAR+ T cells, which did not allow PBZ-induced reactivation and proliferative rescue. The phenomenon, together with the loss of CAR-T target CD19 and the shift of non-CAR CD8+ T cells towards a senescent phenotype likely contributed to set up an immune landscape with poor antitumor capacity.

Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity.

Background: Infusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS. Methods: This is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation. Results: Multivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients. Discussion: Our data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

Mechanisms of Arterial Calcification: The Role of Matrix Vesicles.

Vascular calcification is related to vascular diseases, for example, atherosclerosis, and its comorbidities, such as diabetes and chronic kidney disease. In each condition, a distinctive histological pattern can be recognised that may influence technical choices, possible intra-operative complications, and procedure outcomes, no matter if the intervention is performed by open or endovascular means. This review considers the classification and initiating mechanisms of vascular calcification. Dystrophic and metastatic calcifications, Monckeberg's calcification, and genetic forms are firstly outlined, followed by their alleged initiation mechanisms; these include (a) ineffective macrophage efferocytosis; (b) ectopic osteogenesis driven by modified resident or circulating osteoprogenitors. As in physiological bio-mineralisation, active calcification starts with the deposition of cell derived matrix vesicles into the extracellular matrix. To substantiate this belief, an in depth ultra-structural documentation of hydroxyapatite crystal deposition on such vesicles is provided in an ex-vivo human vascular cell model. Revealing the vesicle composition and phenotype in normal and pathological vascular conditions will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Comparison of Quality of Life in Patients Undergoing Hemodialysis and Peritoneal Dialysis: a Systematic Review and Meta-Analysis.

BACKGROUND/AIMS: The increase in the survival rate of patients with chronic renal failure due to substitution treatment prompts an investigation of their quality of life (QoL), a key measure to evaluate the outcomes of chronic disease treatment. To determine whether hemodialysis or peritoneal dialysis provide a better QoL, a systematic meta-analysis was performed. METHODS: We searched through the database Cinahl, Medline, PubMed, Scopus and Proquest, including articles published from 2011 until June 2016. We selected articles that compared, through KDQOL-SF 1.3 or 36 questionnaires, QoL among patients undergoing hemodialysis and peritoneal dialysis. The data was collected using Excel Office, and t-test has been performed on independent samples to identify significant differences. RESULTS: Only some of the seven articles found significant differences between the two treatments. One of the studies showed a better QoL for peritoneal dialysis patients, while, on the contrary, two other studies support that the best QoL is in patients receiving hemodialysis. Another article displayed significant difference only for satisfaction in relation to care, better in patients on peritoneal dialysis, and for physical health, better in hemodialysis. CONCLUSIONS: The analysis has not led to a unanimous conclusion. Quantitative analysis showed that the only statistically significant difference between the QoL of patients on hemodialysis and peritoneal dialysis regards the effect of kidney disease, which happens to be better in patients undergoing peritoneal dialysis.

A calpain-cleaved fragment of ß-catenin promotes BCRABL1+ cell survival evoked by autophagy induction in response to imatinib.

Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR-ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR-ABL1 tyrosine kinase represents a regulatory mechanism of ß-catenin stability through events encompassing the activation of calpain, which targets ß-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote ß-catenin autolysosomal degradation to originate a regulatory loop where ß-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of ß-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR-ABL1+ cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca(2+) cytosolic concentration and results in the calpain cleavage into a 28kDa fragment implicated in ß-catenin proteasome-independent degradation. More important for BCR-ABL1+ cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of ß-catenin accumulated within the cytoplasmic compartment into a 75kDa fragment, still owning TCF-dependent transcriptional activity. Such a ß-catenin fragment might be crucial for BCR-ABL1+ cell survival following the fusion protein TK inhibition.