Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection.

Introduction: The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. Methods: This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. Results: GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. Discussion: The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings.

Unlocking Predictive Power: Quantitative Assessment of CAR-T Expansion with Digital Droplet Polymerase Chain Reaction (ddPCR).

Flow cytometry (FCM) and quantitative PCR (qPCR) are conventional methods for assessing CAR-T expansion, while digital droplet PCR (ddPCR) is emerging as a promising alternative. We monitored CAR-T transcript expansion in 40 B-NHL patients post-infusion of CAR-T products (axi-cel; tisa-cel; and brexu-cel) with both His-Tag FCM and ddPCR techniques. Sensitivity and predictive capacity for efficacy and safety outcomes of ddPCR were analyzed and compared with FCM. A significant correlation between CAR-T counts determined by FCM and CAR transcripts assessed by ddPCR (p < 0.001) was observed. FCM revealed median CD3+CAR+ cell counts at 7, 14, and 30 days post-infusion with no significant differences. In contrast, ddPCR-measured median copies of CAR-T transcripts demonstrated significant lower copy numbers in tisa-cel recipients compared to the other products at day 7 and day 14. Patients with a peak of CAR transcripts at day 7 exceeding 5000 copies/microg gDNA, termed "good CAR-T expanders", were more likely to achieve a favorable response at 3 months (HR 10.79, 95% CI 1.16-100.42, p = 0.036). Good CAR-T expanders showed superior progression-free survival at 3, 6, and 12 months compared to poor CAR-T expanders (p = 0.088). Those reaching a peak higher than 5000 copies/microg gDNA were more likely to experience severe CRS and ICANS. DdPCR proves to be a practical method for monitoring CAR-T expansion, providing quantitative information that better predicts both treatment outcomes and toxicity.

Hematopoiesis and immune reconstitution after CD19 directed chimeric antigen receptor T-cells (CAR-T): A comprehensive review on incidence, risk factors and current management.

Impaired function of hematopoiesis after treatment with chimeric antigen T-cells (CAR-T) is a frequent finding and can interest a wide range of patients, regardless of age and underlying disease. Trilinear cytopenias, as well as hypogammaglobulinemia, B-cell aplasia, and T-cell impairment, can severely affect the infectious risk of CAR-T recipients, as well as their quality of life. In this review, we provide an overview of defects in hematopoiesis after CAR-T, starting with a summary of different definitions and thresholds. We then move to summarize the main pathogenetic mechanisms of cytopenias, and we offer insight into cytomorphological aspects, the role of clonal hematopoiesis, and the risk of secondary myeloid malignancies. Subsequently, we expose the major findings and reports on T-cell and B-cell quantitative and functional impairment after CAR-T. Finally, we provide an overview of current recommendations and leading experiences regarding the management of cytopenias and defective B- and T-cell function.

The CD4/CD8 ratio of infused CD19-CAR-T is a prognostic factor for efficacy and toxicity.

CD4+ and CD8+ chimeric antigen receptor T cells (CAR-T) play different roles in the in vivo anti-tumour response, but the role of the CD4+ /CD8+ ratio among infused CAR-T has not been clearly defined yet. We analysed leftovers from infused anti-CD19 CAR-T bags of 31 patients with aggressive B-cell lymphomas. The median ratio was 1.44, lower for brexu-cel compared to tisa-cel and axi-cel. The CAR+CD4+ /CD8+ ratio was influenced by lactate dehydrogenase levels at apheresis, not by age, previous treatments or the CD4+ /CD8+ ratio in peripheral blood. Patients with a response at 3 months after CAR-T (M3) had a lower CAR+CD4+ /CD8+ ratio in the infused products compared to non-responders (ratio 0.74 vs. 2.47, p = 0.011). A CAR+CD4+ /CD8+ ratio higher than the cut point of 1.12 was associated with an increased risk of treatment failure at M3 (OR 23.3, p = 0.012) and M6 (OR 10, p = 0.028). The median 6-month PFS was 76% for patients with a ratio lower than 1.12% vs. 31% for the others. The prognostic role of the CAR+CD4+ /CD8+ ratio was independent of the costimulatory domain (CD28 vs. 4-1BB) of the product (OR 16.41, p = 0.041). Our data indicate a crucial role for CD8+ CAR-T and the CAR+CD4+ /CD8+ ratio in predicting CAR-T efficacy.

Endoscopic Ultrasound-Guided Fine Needle Biopsy in the Diagnostic Work-Up of Deep-Seated Lymphadenopathies and Spleen Lesions: A Monocentric Experience.

EUS-FNB has been introduced in clinical practice as a less invasive diagnostic approach with respect to surgery. We performed a single-center retrospective study on the diagnostic efficacy of EUS-guided FNB, including 171 patients with lymph nodes, splenic, and extranodal lesions that underwent EUS for FNB at our institution. Excluding 12 patients who did not undergo FNB and 25 patients with a previous diagnosis of a solid tumor, we included 134 patients with clinical/radiological suspect of a lymphoproliferative disease, including 20 patients with a previous history of lymphoma. Out of the 134 biopsies, material of diagnostic quality was obtained in 111 procedures (84.3%). Histological examination of the EUS-FNB samples produced an actionable diagnosis in 100 cases (74.6%). Among the patients without an actionable diagnosis, a second, different diagnostic procedure produced a further eight diagnoses of lymphoma. Therefore, the sensitivity of EUS-FNB for diagnosing lymphomas was calculated to be 86.4% (51/59). Assignment of lymphomas to WHO classification subtypes was possible in 47/51 (92%) of the cases. In conclusion, EUS-FNB is an effective procedure for the histological characterization of lesions that are suspected to be lymphoproliferative disease, allowing for an actionable diagnosis in 75% of cases.

The Role of Fecal Microbiota Transplantation in the Allogeneic Stem Cell Transplant Setting.

Microbiota changes during allogeneic hematopoietic stem cell transplantation has several known causes: conditioning chemotherapy and radiation, broad-spectrum antibiotic administration, modification in nutrition status and diet, and graft-versus-host disease. This article aims to review the current knowledge about the close link between microbiota and allogeneic stem cell transplantation setting. The PubMed search engine was used to perform this review. We analyzed data on microbiota dysbiosis related to the above-mentioned affecting factors. We also looked at treatments aimed at modifying gut dysbiosis and applications of fecal microbiota transplantation in the allogeneic stem cell transplant field, with particular interest in fecal microbiota transplantation for graft-versus-host disease (GvHD), multidrug-resistant and clostridium difficile infections, and microbiota restoration after chemotherapy and antibiotic therapy.

Discrepancy between recipient and donor rs4364254 heparanase single nucleotide polymorphism impacts graft-versus-host disease after allogeneic stem cell transplant.

INTRODUCTION: The heparanase (HPSE) gene is highly polymorphic, but only a minority of its single nucleotide polymorphisms (SNPs) have been studied. Among these, rs4693608 and rs4364254 SNPs are closely associated with mRNA expression and HPSE protein levels in healthy subjects. Given the association between HPSE and inflammatory response, we aimed to evaluate whether HPSE rs4693608 and rs4364254 SNPs could have an impact on graft-versus-host disease after allogeneic stem cell transplants (HSCT). METHODS: A total of 228 consecutive patients who underwent HSCT at our center between 2005 and 2018 were included. The rs4693608 SNP was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, while the rs4364254 was detected by allele-specific amplification. RESULTS: The recipient-donor discrepancy for rs4364254 HPSE SNP was significantly associated with grade II-IV aGvHD (HR 1.75, p = 0.03). Patients were stratified into risk groups as follows: low-risk group (LDR) including TT-TT, TT-CT, CT-TT, CC-CC; high-risk group (HDR) including CC-CT, CC-TT, CT-CC, CT-CT, TT-CC. Day 100 cumulative incidence of grade II-IV aGvHD was 23.4% in the LDR group and 41.4% in the HDR group (p = 0.01). One-year cumulative incidence of moderate/severe cGvHD was 42.6% in the LDR group and 58.6% in the HDR group (p = 0.04). Independent variables for moderate/severe cGvHD in patients who received myeloablative conditioning included donor rs4693608 SNP (GA/AA vs. GG: HR 6.86, p = 0.008), rs4693608-rs4364254 SNP combination in recipient (HR/MR vs. LR: HR 3.67, p = 0.01), and previous grade II-IV aGvHD (HR 3.28, p = 0.0005). Finally, donors with rs4364254 SNP CC conferred increased transplant-related mortality (TRM) (39.1% vs. 25%, p = 0.03) and decreased graft-relapse free survival (GRFS) (23.5% vs. 34.4%, p = 0.04) compared with CT or TT genotypes. CONCLUSION: The differences in incidence of GvHD according to recipient-donor genotype combinations suggests a possible role for rs4364254 HPSE SNP in predicting GvHD. A high level of HPSE, particularly linked to CC genotype of rs4364254 SNP may promote alloreactive T lymphocytes activation and migration toward target organs.

Consensus for Flow Cytometry Clinical Report on Multiple Myeloma: A Multicenter Harmonization Process Merging Laboratory Experience and Clinical Needs.

Flow cytometry is a highly sensitive and specific approach for discriminating between normal and clonal plasma cells in multiple myeloma. Uniform response criteria after treatment have been established by the International Myeloma Working Group and the EuroFlow Group; however, the way in which flow cytometry data are reported has suffered from no collaborative or multicentre efforts. This study, involving 8 expert laboratories and 12 clinical hematology units of the Lazio region in Italy, aims to produce a uniform and shared report among the various Centres. From the pre-analytical phase to sample processing, data acquisition, analysis, and evaluation of the potential limitations and pitfalls of the entire process, the study reaches a final conclusion shared by laboratories and clinicians according to the most updated principles and recommendations. The aim was to identify the necessary data to be included in the clinical report by using multiple-choice questionnaires at every single stage of the process. An agreement of more than 75% of the laboratories was considered mandatory for the data to be included in the report. By ensuring the operational autonomy of each laboratory, this study provides a clear report that limits subjective interpretations and highlights possible bias in the process, better supporting clinical decision-making.

Evaluation of the Toxic Activity of the Graphene Oxide in the Ex Vivo Model of Human PBMC Infection with Mycobacterium tuberculosis.

Graphene Oxide has been proposed as a potential adjuvant to develop improved anti-TB treatment, thanks to its activity in entrapping mycobacteria in the extracellular compartment limiting their entry in macrophages. Indeed, when administered together with linezolid, Graphene Oxide significantly enhanced bacterial killing due to the increased production of Reactive Oxygen Species. In this work, we evaluated Graphene Oxide toxicity and its anti-mycobacterial activity on human peripheral blood mononuclear cells. Our data show that Graphene Oxide, different to what is observed in macrophages, does not support the clearance of Mycobacterium tuberculosis in human immune primary cells, probably due to the toxic effects of the nano-material on monocytes and CD4+ lymphocytes, which we measured by cytometry. These findings highlight the need to test GO and other carbon-based nanomaterials in relevant in vitro models to assess the cytotoxic activity while measuring antimicrobial potential.

Pre-transplant gene profiling characterization by next-generation DNA sequencing might predict relapse occurrence after hematopoietic stem cell transplantation in patients affected by AML.

Background: In the last decade, many steps forward have been made in acute myeloid leukemia prognostic stratification, adding next-generation sequencing techniques to the conventional molecular assays. This resulted in the revision of the current risk classification and the introduction of new target therapies. Aims and methods: We wanted to evaluate the prognostic impact of acute myeloid leukemia (AML) mutational pattern on relapse occurrence and survival after allogeneic stem cell transplantation. A specific next-generation sequencing (NGS) panel containing 26 genes was designed for the study. Ninety-six patients studied with NGS at diagnosis were included and retrospectively studied for post-transplant outcomes. Results: Only eight patients did not show any mutations. Multivariate Cox regression revealed FLT3 (HR, 3.36; p=0.02), NRAS (HR, 4.78; p=0.01), TP53 (HR, 4.34; p=0.03), and WT1 (HR 5.97; p=0.005) mutations as predictive variables for relapse occurrence after transplantation. Other independent variables for relapse recurrence were donor age (HR, 0.97; p=0.04), the presence of an adverse cytogenetic risk at diagnosis (HR, 3.03; p=0.04), and the obtainment of complete remission of the disease before transplantation (HR, 0.23; p=0.001). Overall survival appeared to be affected only by grade 2-4 acute GvHD occurrence (HR, 2.29; p=0.05) and relapse occurrence (HR, 4.33; p=0.0001) in multivariate analysis. Conclusions: The small number of patients and the retrospective design of the study might affect the resonance of our data. Although results on TP53, FLT3, and WT1 were comparable to previous reports, the interesting data on NRAS deserve attention.

Multicolor flow cytometry on pericardial effusion for a prompt diagnosis and treatment of hematological malignancies with heart involvement.

Background: Malignancies represent 15-50% of total causes of pericardial effusions (PE). Routine analyses recommended to be performed on pericardial fluid include general chemistry, cytology, polymerase chain reaction, and microbiological cultures. Multicolor flow cytometry (FC) is a laboratory test that already proved to be useful in the detection of lymphoproliferative and metastatic malignancies in pleural and peritoneal effusions, but current guidelines do not mention its use on PE to reach a diagnosis. Methods: Our institutional protocol foresees to routinely perform a multicolor FC analysis on pericardial fluid samples obtained by pericardiocentesis, in addition to other guidelines-recommended analyses. A sample of 15-30 ml is analyzed using a lyse and wash staining method using combination panels of antibodies, allowing to detect specific cellular subpopulations, analyzing tens to hundreds of thousands of cells in few seconds. The present manuscript aims to report our single-center experience with this diagnostic tool in patients presenting with PE requiring pericardiocentesis. Results: Routine use of multicolor FC on pericardial fluid samples in our institution allowed to reach a definite diagnosis of cardiac lymphomas in two patients presenting with otherwise unexplained severe PE. This resulted in immediate start of combined immunotherapy, with patients' clinical improvement. At 6 months follow-up both patients are alive and presented a complete disease regression. Conclusion: Preliminary evidence from routine use of multicolor FC on PE support that this is a promising tool to reach a rapid diagnosis of hematological malignancies with heart involvement, leading to a prompt initiation of targeted therapies.

Day +60 WT1 assessment on CD34 selected bone marrow better predicts relapse and mortality after allogeneic stem cell transplantation in acute myeloid leukemia patients.

The aim of this study was to evaluate the role of WT1 expression after allogeneic stem cell transplantation (alloHSCT) in patients with acute myeloid leukemia (AML). We studied WT1 expression in bone marrow cells from 50 patients in complete remission on day +60 after transplant. WT1 was assessed on unfractionated bone marrow mononuclear cells (MNC) and on CD34+ selected cells (CD34+). A ROC curve analysis identified 800 WT1 copies on CD34+ selected cells, as the best cut-off predicting relapse (AUC 0.842, p=0.0006, 85.7% sensitivity and 81.6% specificity) and 100 copies in MNC (AUC 0.819, p=0.007, 83.3% sensitivity and 88.2% specificity). Using the 800 WT1 copy cut off in CD34+ cells, the 2 year cumulative incidence of relapse was 12% vs 38% (p=0.005), and 2 year survival 88% vs 55% (p=0.02). Using the 100 WT1 copy cut off in unfractionated MNC, the 2 year cumulative incidence of relapse 13% vs 44% (p=0.01) and the 2 year survival 88% vs 55% (p=0.08). In a multivariate Cox analysis WT1 expression in CD34 cells proved to highly predictive of relapse (p=0.004); also WT1 expression on unfractionated cells predicted relapse (p=0.03). In conclusion, day-60 WT1 expression after allogeneic HSCT is a significant predictor of relapse, particularly when tested on CD34+ selected bone marrow cells.

PD-L1 expression in peripheral blood granulocytes at diagnosis as prognostic factor in classical Hodgkin lymphoma.

Hodgkin lymphoma (HL) is a neoplastic disease in which the inflammatory microenvironment plays a pivotal role in the tumorigenesis. Neutrophilia is a typical finding in HL at diagnosis and, in particular, in association with lymphocytopenia, is a negative prognostic factor. As the immune checkpoint Programmed Death (PD)-L1/PD-1 has become an important therapeutic target, we were interested in the expression of PD-L1 in peripheral blood (PB) leukocytes using flow cytometry and RT-PCR in patients with HL and healthy controls. Granulocytes were the major PB cell fraction expressing PD-L1. PD-L1 expression on granulocytes was higher in patients with HL than in controls and correlated with lower T-cell numbers in PB. We analyzed for associations between PD-L1 expression in PB granulocytes at the time of diagnosis with patient characteristics and outcome in 126 patients with HL treated with standard chemotherapy adriamycin, bleomycin, vinblastine, and dacarbazine. Increased PD-L1 expression in PB associated with advanced disease, systemic symptoms, positive interim positron emission tomography, and inferior progression-free survival (PFS). PFS at 4 years was 81% (95% C.I., 71-87%) in patients with normal PD-L1 expression and 56% (95% C.I., 35-72%) in patients with higher-than-normal PD-L1 expression (p = 0.002). In conclusion, PD-L1 expression in PB could become a potentially actionable prognostic factor in HL.

Expression of PD-L1 in peripheral blood reflects disease burden and predicts interim PET result and prognosis in classical HL.