Early CAR- CD4+ T-lymphocytes recovery following CAR-T cell infusion: A worse outcome in diffuse large B cell lymphoma.

CAR- CD4+ T cell lymphopenia is an emerging issue following CAR-T cell therapy. We analyzed the determinants of CD4+ T cell recovery and a possible association with survival in 31 consecutive patients treated with commercial CAR-T for diffuse large B-cell (DLBCL) or mantle cell lymphoma. Circulating immune subpopulations were characterized through multiparametric-flow cytometry. Six-month cumulative incidence of CAR- CD4+ T cell recovery (≥200 cells/µL) was 0.43 (95% confidence interval [CI]: 0.28-0.65). Among possible determinants of CD4+ T cell recovery, we recognized infusion of a 4-1BB product (tisagenlecleucel, TSA) in comparison with a CD28 (axicabtagene/brexucabtagene, AXI/BRX) (hazard ratio [HR] [95% CI]: 5.79 [1.16-24.12] p = 0.016). Higher CD4+ T cell counts resulted with TSA at month-1, -2 and -3. Moderate-to-severe infections were registered with prolonged CD4+ T cell lymphopenia. Early, month-1 CD4+ T cell recovery was associated with a worse outcome in the DLBCL cohort, upheld in a multivariate regression model for overall survival (HR: 4.46 [95% CI: 1.12-17.71], p = 0.03). We conclude that a faster CAR- CD4+ T cell recovery is associated with TSA as compared to AXI/BRX. Month-1 CAR- CD4+ T cell subset recovery could represent a "red flag" for CAR-T cell therapy failure in DLBCL patients.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

Pre-transplant minimal residual disease assessment and transplant-related factors predict the outcome of acute myeloid leukemia patients undergoing allogeneic stem cell transplantation.

We studied pretransplant minimal residual disease (MRD) in 224 patients (median age 44 years; range 17-65) with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplant (HSCT) in complete remission. MRD was evaluated on marrow samples using multicolor flow cytometry and assessment of WT1 gene expression. Both methods showed a strong prognostic value and their combination allowed the identification of three groups of patients with different risk of relapse. In multivariate analysis, combined MRD was the only predictor of cumulative incidence of relapse, regardless of donor type, conditioning regimen, first or second CR at HSCT, HSCT year, and ELN risk group. Multivariate regression model showed that only negative combined MRD status (P < .001) and myeloablative conditioning (P = .004) were independently associated with better OS. Among MRD-positive patients, a reduced incidence of relapse was observed in patients receiving haplo transplant (P < .05) and in patients who showed grade II-IV aGVHD (P < .03). In patients with negative combined MRD, the intensity of conditioning regimen did not affect the overall favorable outcome. We suggest that pretransplant MRD evaluation combined with transplant-related factors can identify AML patients at higher risk for relapse and might help in defining the overall transplant strategy.

Bronchoalveolar lavage fluid characteristics and outcomes of invasively mechanically ventilated patients with COVID-19 pneumonia in Genoa, Italy.

BACKGROUND: The primary objective of the study is to describe the cellular characteristics of bronchoalveolar lavage fluid (BALF) of COVID-19 patients requiring invasive mechanical ventilation; the secondary outcome is to describe BALF findings between survivors vs non-survivors. MATERIALS AND METHODS: Patients positive for SARS-CoV-2 RT PCR, admitted to ICU between March and April 2020 were enrolled. At ICU admission, BALF were analyzed by flow cytometry. Univariate, multivariate and Spearman correlation analyses were performed. RESULTS: Sixty-four patients were enrolled, median age of 64 years (IQR 58-69). The majority cells in the BALF were neutrophils (70%, IQR 37.5-90.5) and macrophages (27%, IQR 7-49) while a minority were lymphocytes, 1%, TCD3+ 92% (IQR 82-95). The ICU mortality was 32.8%. Non-survivors had a significantly older age (p = 0.033) and peripheral lymphocytes (p = 0.012) were lower compared to the survivors. At multivariate analysis the percentage of macrophages in the BALF correlated with poor outcome (OR 1.336, CI95% 1.014-1.759, p = 0.039). CONCLUSIONS: In critically ill patients, BALF cellularity is mainly composed of neutrophils and macrophages. The macrophages percentage in the BALF at ICU admittance correlated with higher ICU mortality. The lack of lymphocytes in BALF could partly explain a reduced anti-viral response.

Prognostic relevance of a blastic plasmacytoid dendritic cell neoplasm-like immunophenotype in cytogenetically normal acute myeloid leukemia patients.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a poor prognosis myeloid malignancy characterized by an atypical phenotype (CD123+, CD56+, and CD4+). We reported that BPDCN-like phenotype (CD123+ and either CD56+ or CD4+ or both) confers poor prognosis to acute myeloblastic leukemia (AML) patients with mutated NPM1. Here, we evaluated the incidence and the prognostic relevance of BPDCN-like phenotype in cytogenetically normal AML (CN-AML) patients. From 2006 to 2016, 83 young (age <60 yrs), consecutive, CN-AML patients underwent intensive treatment. Fifteen patients (18%) showed a BPDCN-like phenotype with no difference between NPM1-mutated (mut) and NPM1-wt patients. It did not significantly affect survival neither in the whole cohort, nor in NPM1-wt patients. However, as reported, it conferred a dismal prognosis in NPM1-mut AML (p < 0.001), irrespectively of the mutational status for FLT3-ITD. In conclusion we show that BPDCN-like phenotype displays a negative prognostic relevance only in NPM1-mutated AML.

A simple cytofluorimetric score may optimize testing for biallelic CEBPA mutations in patients with acute myeloid leukemia.

Acute myeloid leukemia with biallelic mutation of CEBPA (CEBPA-dm AML) is a distinct good prognosis entity recognized by WHO 2016 classification. However, testing for CEBPA mutation is challenging, due to the intrinsic characteristics of the mutation itself. Indeed, molecular analysis cannot be performed with NGS technique and requires Sanger sequencing. The association of recurrent mutations or translocations with specific immunophenotypic patterns has been already reported in other AML subtypes. The aim of this study was the development of a specific cytofluorimetric score (CEBPA-dm score), in order to distinguish patients who are unlikely to harbor the mutation. To this end, the correlation of CEBPA-dm score with the presence of the mutation was analyzed in 50 consecutive AML patients with normal karyotype and without NPM1 mutation (that is mutually exclusive with CEBPA mutation). One point each was assigned for expression of HLA DR, CD7, CD13, CD15, CD33, CD34 and one point for lack of expression of CD14. OS was not influenced by sex, age and CEBPA-dm score. Multivariate OS analysis showed that CEBPA-dm (p < 0.02) and FLT3-ITD (p < 0.01) were the strongest independent predictors of OS. With a high negative predictive value (100%), CEBPA-dm score < 6 was able to identify patients who are unlikely to have the mutation. Therefore, the application of this simple score might optimize the use of expensive and time-consuming diagnostic and prognostic assessment in the baseline work up of AML patients.

Geena 2, improved automated analysis of MALDI/TOF mass spectra.

BACKGROUND: Mass spectrometry (MS) is producing high volumes of data supporting oncological sciences, especially for translational research. Most of related elaborations can be carried out by combining existing tools at different levels, but little is currently available for the automation of the fundamental steps. For the analysis of MALDI/TOF spectra, a number of pre-processing steps are required, including joining of isotopic abundances for a given molecular species, normalization of signals against an internal standard, background noise removal, averaging multiple spectra from the same sample, and aligning spectra from different samples. In this paper, we present Geena 2, a public software tool for the automated execution of these pre-processing steps for MALDI/TOF spectra. RESULTS: Geena 2 has been developed in a Linux-Apache-MySQL-PHP web development environment, with scripts in PHP and Perl. Input and output are managed as simple formats that can be consumed by any database system and spreadsheet software. Input data may also be stored in a MySQL database. Processing methods are based on original heuristic algorithms which are introduced in the paper. Three simple and intuitive web interfaces are available: the Standard Search Interface, which allows a complete control over all parameters, the Bright Search Interface, which leaves to the user the possibility to tune parameters for alignment of spectra, and the Quick Search Interface, which limits the number of parameters to a minimum by using default values for the majority of parameters. Geena 2 has been utilized, in conjunction with a statistical analysis tool, in three published experimental works: a proteomic study on the effects of long-term cryopreservation on the low molecular weight fraction of serum proteome, and two retrospective serum proteomic studies, one on the risk of developing breat cancer in patients affected by gross cystic disease of the breast (GCDB) and the other for the identification of a predictor of breast cancer mortality following breast cancer surgery, whose results were validated by ELISA, a completely alternative method. CONCLUSIONS: Geena 2 is a public tool for the automated pre-processing of MS data originated by MALDI/TOF instruments, with a simple and intuitive web interface. It is now under active development for the inclusion of further filtering options and for the adoption of standard formats for MS spectra.

Complement C3f serum levels may predict breast cancer risk in women with gross cystic disease of the breast.

Gross cystic disease (GCDB) is a breast benign condition predisposing to breast cancer. Cryopreserved sera from GCDB patients, some of whom later developed a cancer (cases), were studied to identify potential risk markers. A MALDI-TOF mass spectrometry analysis found several complement C3f fragments having a significant increased abundance in cases compared to controls. After multivariate analysis, the full-length form of C3f maintained a predictive value of breast cancer risk. Higher levels of C3f in the serum of women affected by a benign condition like GCDB thus appears to be correlated to the development of breast cancer even 20 years later. BIOLOGICAL SIGNIFICANCE: Increased complement system activation has been found in the sera of women affected by GCDB who developed a breast cancer, even twenty or more years later. C3f may predict an increased breast cancer risk in the healthy population and in women affected by predisposing conditions.

Dihydrotestosterone and bicalutamide do not affect periostin expression in androgen-dependent LNCaP prostate cancer cell lines.

BACKGROUND/AIM: To investigate periostin (POSTN) expression in the LNCaP cell line. MATERIALS AND METHODS: Our LNCaP strain did not constitutively express the POSTN gene. Through cell transfection with a cloning vector, we developed an LNCaP cell line that stably expressed POSTN. LNCaP wild-type and transfected cells were incubated with dihydrotestosterone (DHT) in the presence/or absence of bicalutamide (BIC). POSTN mRNA was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and growth was measured with the MTT assay. RESULTS: POSTN transfection stimulated LNCaP cell growth. While POSTN transfection did not interfere with the stimulatory effect of DHT, BIC had an inhibitory effect on cell proliferation. However, exposure to either DHT and/or BIC was not able to interfere with POSTN expression per se. CONCLUSION: We confirmed the role of POSTN in promoting cancer cell growth. Although POSTN transcription is not likely to be androgen-dependent, the fact that increased cell proliferation POSTN-mediated was impaired by BIC suggests an androgen modulation of POSTN interaction proteins.

Effects of bicalutamide and 4OH-tamoxifen on androgen-regulated gene expression in the LNCaP cell line.

BACKGROUND: Bicalutamide (BIC) is an alternative treatment to castration for advanced prostate cancer. Breast events are common adverse effects which can be effectively prevented by the concurrent administration of tamoxifen, a selective estrogen receptor modulator. MATERIALS AND METHODS: We investigated the effects of BIC, 4-hydroxy Tamoxifen (4OHT), the active metabolite of tamoxifen, and their combination on the expression of a panel of genes implicated in prostate cancer development and progression in LNCaP cells stimulated with dihydrotestosterone. RESULTS: Our findings confirm the anti-proliferative activity of BIC on LNCaP cell growth but also show the down-regulating function of this anti-androgen on the expression of genes involved in tumor proliferation and invasion [cyclins, caspases, epidermal growth factor (EGF)]. The combination with 4OHT exerts a synergistic effect on the downregulation of some genes involved in prostate cancer progression. CONCLUSION: The observation that the expression of several genes [such as B-cell lymphoma-2 (BCL2), myelocytomatosis oncogene (MYC), caspases] is modulated midly-to-moderately, after 4OHT addition suggests that this combined approach in the clinical setting should be further investigated through appropriate trials.

Prognostic value of nuclear matrix protein expression in localized prostate cancer.

PURPOSE: The aim of the study was to correlate nuclear matrix (NM) protein expression profiles with the risk of PSA progression or death in early prostate cancer (PCa). METHODS: High-resolution two-dimensional gel electrophoresis (2D-PAGE) was used to identify tumor-associated NM proteins in the PCa specimens obtained from 94 patients. The association between the expression of each protein and the probability of PSA progression or death was studied through univariate analysis. Unsupervised hierarchical clustering analysis was then used to generate patient clusters showing comparable outcomes by including the proteins that were predictive at univariate analysis. PSA-free and overall survival curves relative to each cluster were constructed by means of the Kaplan-Meier method and curves compared by the log-rank test. Multi-parametric models were constructed according to Cox proportional hazard technique. RESULTS: After a median follow-up of 11.7 years (range, 6.5-16.2), 50 patients progressed and 22 died. Of the eight NM proteins identified through 2D-PAGE, proteins NM-6, NM-7 and NM-8 were confirmed to be individually associated with a higher risk of PSA progression at univariate analysis. Proteins NM-6 and NM-8 were also predictive of survival probability. Hierarchical clustering analysis of these proteins allowed to identify one cluster of tumors co-expressing the three proteins or proteins NM-6 and NM-8, characterized by a very poor outcome, suggesting a specific role for these proteins in PCa progression. The predictive value of this mini-signature in respect to PSA-free survival was confirmed by multivariate analysis. CONCLUSIONS: Changes in NM scaffolding are strongly associated with the clinical outcome of patients following radical prostatectomy.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome.

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

Mutant presenilin 1 increases the expression and activity of BACE1.

Mutations of the presenilin 1 (PS1) gene are the most common cause of early onset familial Alzheimer disease (FAD). PS1 mutations alter the activity of the gamma-secretase on the beta-amyloid precursor protein (APP), leading to selective overproduction of beta-amyloid (Abeta) 42 peptides, the species that forms oligomers that may exert toxic effects on neurons. Here we show that PS1 mutations, expressed both transiently and stably, in non-neuronal and neuronal cell lines increase the expression and the activity of the beta-secretase (BACE1), the rate-limiting step of Abeta production. Also, BACE1 expression and activity are elevated in brains of PS1 mutant knock-in mice compared with wild type littermates as well as in cerebral cortex of FAD cases bearing various PS1 mutations compared with in sporadic AD cases and controls. The up-regulation of BACE1 by PS1 mutations requires the gamma-secretase cleavage of APP and is proportional to the amount of secreted Abeta42. Abeta42, and not AICD (APP intracellular domain), is indeed the APP derivative that mediates the overexpression of BACE1. The effect of PS1 mutations on BACE1 may contribute to determine the wide clinical and pathological phenotype of early onset FAD.

Accelerated senescence in the kidneys of patients with type 2 diabetic nephropathy.

We examined the hypothesis that senescence represents a proximate mechanism by which the kidney is damaged in type 2 diabetic nephropathy (DN). As a first step, we studied whether the senescence-associated beta-galactosidase (SA-beta-Gal) and the cell cycle inhibitor p16INK4A are induced in renal biopsies from patients with type 2 DN. SA-beta-Gal staining was approximately threefold higher (P < 0.05) than in controls in the tubular compartment of diabetic kidneys and correlated directly with body mass index and blood glucose. P16INK4A expression was significantly increased in tubules (P < 0.005) and in podocytes (P = 0.04). Nuclear p16INK4A in glomeruli was associated with proteinuria (P < 0.002), while tubular p16INK4A was directly associated with body mass index, LDL cholesterol, and HbA1c (P < 0.001-0.05). In a parallel set of experiments, proximal tubule cells passaged under high glucose presented a limited life span and an approximately twofold increase in SA-beta-Gal and p16INK4A protein. Mean telomere lengths decreased approximately 20% as an effect of replicative senescence. In addition, mean telomere decreased further by approximately 30% in cells cultivated under high glucose. Our results show that the kidney with type 2 diabetic nephropathy displays an accelerated senescent phenotype in defined renal cell types, mainly tubule cells and, to a lesser extent, podocytes. A similar senescent pattern was observed when proximal tubule cell cultures where incubated under high-glucose media. These changes are associated with shortening tubular telomere length in vitro. These findings indicate that diabetes may boost common pathways involving kidney cell senescence, thus reinforcing the role of the metabolic syndrome on biological aging of tissues.

Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres.

Summary The purpose of this study was to evaluate telomere length in peripheral blood granulocytes and mononuclear cells collected from 22 women with polycythaemia vera (PV) and essential thrombocythaemia (ET). PV and ET are chronic myeloproliferative diseases whose heterogeneity of stem cell origin and clonal development has been established through analysis of X-chromosome inactivation patterns. The results from clonality assay and determination of telomere length show that only clonal granulocytes have shortened telomeres.

High telomerase activity in granulocytes from clonal polycythemia vera and essential thrombocythemia.

Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative disorders that share the involvement of a multipotent progenitor cell and dominance of the transformed clone over normal hematopoiesis. On the other hand, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been well established. To identify useful prognostic indicators, we analyzed telomerase activity (TA), a known marker of neoplastic proliferation, in granulocytes (PMNs) and mononuclear cells (MNCs) from 22 female patients with ET and PV. Clonality status was determined by investigation of X chromosome inactivation patterns (XCIPs). We found a statistically significant positive correlation between high TA and monoclonal pattern of XCIP. Therefore, our data suggest that the use of multiple tumor markers may contribute to a better understanding of the deregulated physiology of these disorders and provide useful prognostic factors.

Influenza vaccine in chronic lymphoproliferative disorders and multiple myeloma.

OBJECTIVE: Vaccination against influenza in patients with chronic lymphoproliferative disorders (CLPD) and multiple myeloma (MM) is still a matter of clinical uncertainty. The aim of this study was to determine the safety, immunogenicity and clinical response to a commercially available vaccine against influenza in a group of such patients. METHODS: Thirty-four patients with CLPD and MM and 34 immunologically normal subjects were vaccinated with the same vaccine against influenza. Patients were observed during the epidemic season from October 1999 to April 2000, and monitored for side-effects of the vaccine, seroprotection and seroconversion after vaccination. The prevaccination level of immunoglobulins was also determined. Occurrence of influenza episodes was demonstrated with the positive isolation of a viral strain from a pharyngeal swab. RESULTS: No patient had untoward reactions to the vaccine used. Seroconversion and seroprotection were up to the standard established by the European Agency for the Evaluation of Medicinal Products. Only one patient developed influenza during follow-up. CONCLUSIONS: Influenza vaccine is effective and well tolerated in patients with CLPD and MM. No contraindications exist for its use, and it should become a routine practice, in order to prevent serious complications during the influenza epidemic season.

Exposure of erythrocytes to methylene blue shows the active role of catalase in removing hydrogen peroxide.

Methylene blue (MB) is a powerful reducing agent that is widely used in clinical practice as well as for metabolic studies of the erythrocyte. We have investigated the role of catalase as a specific enzyme for the removal of hydrogen peroxide by measuring the in vitro effects of MB on human red cells. In the presence of MB, catalase underwent inactivation even with the co-existence of active generation of NADPH, leaving the glutathione concentration unaffected. The data obtained in the present investigation show, using a different tool (MB), that catalase is the active enzyme in H2O2 detoxification and that its integrity is largely dependent on an adequate generation of NADPH.