Chronic lymphocytic leukemia and mantle cell lymphoma: similarities and differences from an integrated biological and clinical approach

Introduzione. La leucemia linfatica cronica (CLL) e il linfoma a cellule del mantello (MCL) sono malattie linfoproliferative non guaribili con decorso clinico eterogeneo. Oltre al riconoscimento della loro eterogeneità molecolare, sta emergendo un crescente interesse per le somiglianze tra CLL e MCL. Sebbene il MCL sia una malattia aggressiva, un suo sottotipo presenta un decorso clinico indolente, ed è stato recentemente definito dalla World Health Organization (WHO) sottotipo non-nodal o CLL-like. Il riconoscimento dei parallelismi tra CLL e MCL - in particolare la centralità della via del B-cell receptor (BCR) - è stato il punto di partenza del nostro lavoro. Obiettivi. Indagare i meccanismi alla base della diversa attivazione della via del BCR nel MCL, rispetto alla CLL, e individuare relazioni con l’outcome clinico. Metodi. Abbiamo applicato la citometria a flusso fosfo-specifica combinata con il fluorescent cell barcoding (FCB) a linee cellulari di MCL e a cellule di pazienti affetti da MCL. Abbiamo misurato lo stato di fosforilazione di nove fosfoproteine della via del BCR, ossia pSYK, pLCK, pBTK, pPLCγ2, pp38, pERK1/2, pAKT, pNF-κB p65, pSTAT5, in cellule mononucleate di sangue periferico di 10 pazienti con MCL, raccolte alla diagnosi. Lo stato di fosforilazione delle proteine è stato misurato nella condizione basale e dopo stimolazione del BCR con anticorpi anti-IgG, anti-IgD, anti-IgM o la loro combinazione. I valori di intensità di fluorescenza sono stati espressi come inverse hyperbolic sine (arcsinh) e corretti per i segnali di autofluorescenza. Le risposte alla stimolazione del BCR sono state calcolate rispetto ai segnali nella condizione basale. I dati sono stati sottoposti a una unsupervised hierarchical cluster analysis (HCA) tra i campioni di MCL. L'associazione tra gruppi di pazienti individuati sulla base delle differenze nella via del BCR e parametri clinici è stata calcolata con il Fisher’s exact test. Infine, abbiamo effettuato un confronto esplorativo tra i dati ottenuti nei casi di MCL e quelli ottenuti in 10 pazienti con CLL. Risultati. Nei pazienti con MCL, l'analisi delle fosfoproteine nella condizione basale ha rivelato livelli elevati di pBTK, pPLCγ2 e pSTAT5, con livelli basali nulli o molto bassi di pSYK, pLCK, pAKT e pNF-κB p65. Dopo stimolazione del BCR con anti-IgM, abbiamo dimostrato un livello più elevato sia dei nodi prossimali (pSYK, pLCK, pBTK, pPLCγ2) sia di quelli più distali (pp38, pERK1/2, pAKT) rispetto al basale, ad eccezione di pNF-κB p65 e pSTAT5. Al contrario, la stimolazione con anti-IgG o anti-IgD non ha indotto l'attivazione delle fosfoproteine, ad eccezione di pAKT, che è risultata significativamente attivata dopo la stimolazione con anti-IgG. La unsupervised HCA ha identificato due gruppi di pazienti con MCL: il cluster 1 comprendeva campioni con una risposta di attivazione inferiore nella via del BCR, mentre il cluster 2 includeva campioni con una risposta più elevata. Il raggruppamento dei pazienti basato sul segnale del BCR è risultato significativamente associato ai parametri clinici quali età, risposta alla terapia e POD (progression of disease) precoce o tardiva. Infine, abbiamo dimostrato una netta divisione tra i pazienti affetti da CLL e MCL, confermando così che questa analisi è in grado di distinguere le due diverse popolazioni di pazienti. Conclusioni. FCB e citometria a flusso fosfo-specifica sono versatili ed efficienti e ci hanno permesso di ottenere risultati significativi sui campioni di pazienti. Le perturbazioni di segnale a livello di singola cellula hanno fornito informazioni complementari alle informazioni biologiche e cliniche disponibili per i pazienti con MCL e CLL. Questa metodica potrebbe diventare un test prognostico/predittivo clinicamente utile, dopo validazione in serie indipendenti di pazienti. I nostri risultati rappresentano una premessa per un approccio biologico e clinico integrato alla CLL e al MCL.Introduction. Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are incurable lymphoid malignancies with a highly heterogeneous clinical course. Along with their molecular heterogeneity, growing interest is emerging in CLL and MCL similarities. Although MCL is generally an aggressive disease, a subset of patients shows an indolent clinical course, recently described by the World Health Organization (WHO) has non-nodal or CLL-like MCL. The recognition of the numerous parallels between CLL and MCL - in particular the centrality of the B-cell receptor (BCR) signaling pathway - was the starting point of our research. Aims. To explore the mechanisms behind variability in BCR signaling capacity in MCL, as compared to CLL, and to unveil possible relationship with clinical outcome. Methods. We experimentally applied phospho-specific flow cytometry combined with fluorescent cell barcoding (FCB) to MCL cell lines and patients. Using this technique, we measured the phosphorylation status of nine BCR signaling phosphoproteins, namely pSYK, pLCK, pBTK, pPLCg2, pp38, pERK1/2, pAKT, pNF-kB p65, pSTAT5, in peripheral blood mononuclear cells (PBMCs) from 10 MCL patients collected at diagnosis. The phosphorylation status of signaling proteins was measured in the basal condition and following BCR stimulation with anti-IgG, anti-IgD, anti-IgM antibodies, or their combination. Fluorescence intensity values were expressed as inverse hyperbolic sine (arcsinh) and corrected for the autofluorescence signals. Responses to BCR stimulation were calculated relative to signals in the basal condition. Then, arcsinh transformed data were subjected to unsupervised hierarchical cluster analysis (HCA) within the MCL samples. Association between signaling-defined groups of patients and clinical parameters was performed using Fisher’s exact test. Finally, we made some explorative comparison between data obtained on MCL and 10 CLL patients. Results. In MCL patients, the analysis of signaling phosphoproteins in the basal condition revealed high levels of pBTK, pPLCg2, and pSTAT5, with no or very low basal levels of pSYK, pLCK, pAKT and pNF-kB p65. After BCR stimulation with anti-IgM, we showed an overall significant higher level of both BCR proximal (pSYK, pLCK, pBTK, pPLCg2) and more distal (pp38, pERK1/2, pAKT) signaling nodes compared to unstimulated, excepted for pNF-kB p65 and pSTAT5. In contrast, anti-IgG or anti-IgD stimulation did not induce phosphoproteins activation, except for pAKT, which was significantly activated after anti-IgG stimulation. Unsupervised HCA identified two main clusters of MCL patients: cluster 1 comprised samples with a lower signaling response, whereas cluster 2 included samples showing a higher signaling response. Remarkably, the BCR signaling-based clustering was significantly associated with clinical parameters including age, response to therapy, and early or late-POD (progression of disease). Finally, we demonstrated a clear clustering dividing CLL and MCL patients, thus confirming that this analysis was able to distinguish the two different patients’ populations. Conclusion. FCB and phospho-specific flow cytometry proved versatile and efficient and allowed us to obtain reproducible and significant results on patients’ samples. Signaling perturbations at the single cell level provided complementary information to the available biological and clinical information in MCL and CLL patients. This tool may be candidate to become a clinically useful prognostic/predictive test in the future, after it has been validated in independent series. Our results represent a premise for an integrated biological and clinical approach to CLL and MCL

Prognostic impact of left ventricular mass severity according to the classification proposed by the American society of echocardiography/European association of echocardiography

Background: The American Society of Echocardiography (ASE) and European Association of Echocardiography (EAE) recommend the use of quantitative estimation of left ventricular (LV) mass and defined partition values for mild, moderate, and severe hypertrophy. However, the prognostic implications associated with this categorization are unknown. Methods: In this observational cohort study of unselected adults undergoing echocardiography for any indication, LV hypertrophy was assessed using the ASE/EAE-recommended formula and measurement convention from LV linear dimensions indexed to body surface area. Mortality and incident hospitalizations for cardiovascular disease were the outcomes of this study. Results: Of 2,545 subjects (mean age, 61.9 ± 15.8 years; 56.3% women), 52.9% had normal LV mass, and 15.4% had mild, 12.1% moderate, and 19.6% severe LV hypertrophy. During a mean follow-up period of 2.5 ± 1.2 years, 121 deaths and 292 incident hospitalizations for cardiovascular disease occurred. In multivariate models including age, gender, LV ejection fraction, wall motion score index, significant valvular disease, and atrial fibrillation, the adjusted hazard ratios for death were 1.81 (95% confidence interval [CI], 1.03-3.20; P =.041) for mild, 2.31 (95% CI, 1.33-4.01; P =.003) for moderate, and 2.30 (95% CI, 1.39-3.79, P =.001) for severe LV hypertrophy. The adjusted hazard ratios for incident cardiovascular hospitalizations were 1.24 (95% CI, 0.84-1.82; P =.277) for mild, 2.02 (95% CI, 1.42-2.88; P =.0001) for moderate, and 2.38 (95% CI, 1.75-3.22, P <.0001) for severe LV hypertrophy. After adjustment for known risk predictors, there was a 1.3-fold risk for death and cardiovascular disease events per category of LV mass (P =.001). Conclusions: In a cohort study of unselected adult outpatients, the categorization of LV mass according to the ASE/EAE recommendations offered prognostic information independently of age, gender, and other known predictors. Copyright 2011 by the American Society of Echocardiography

Oncogenic Mutations of MYD88 and CD79B in Diffuse Large B-Cell Lymphoma and Implications for Clinical Practice

Simple SummaryA diagnosis of diffuse large B-cell lymphoma in our therapeutic era should be implemented by the definition of the cell of origin, additional immunohistochemistry (i.e., BCL2 and MYC), and by fluorescent in-situ hybridization. The next step, suggested by the seminary works we will discuss in this review, will be to implement the definition of sub-categories by the recognition of single gene mutations and pathways that may be targetable by newer drugs. We here describe the impact that MYD88 and CD79B activating mutations, two of the most frequent mutations in several DLBCL subtypes, may achieve in the next future in the diagnosis and therapeutics of such a relevant lymphoma subtype.Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma in adults. Despite the recognition of transcriptional subtypes with distinct functional characteristics, patient outcomes have not been substantially altered since the advent of chemoimmunotherapy (CIT) twenty years ago. Recently, a few pivotal studies added to the disease heterogeneity by describing several activating mutations, which have been associated with disease presentation, B-cell function and behavior, and final outcome. DLBCL arises from antigen exposed B-cells, with the B-cell receptor (BCR) playing a central role. BCR-activity related mutations, such as CD79B and MYD88, are responsible for chronic activation of the BCR in a substantial subset of patients. These mutations, often coexisting in the same patient, have been found in a substantial subset of patients with immune-privileged (IP) sites DLBCLs, and are drivers of lymphoma development conferring tissue-specific homing properties. Both mutations have been associated with disease behavior, including tumor response either to CIT or to BCR-targeted therapy. The recognition of CD79B and MYD88 mutations will contribute to the heterogeneity of the disease, both in recognizing the BCR as a potential therapeutic target and in providing genetic tools for personalized treatment

Bendamustine plus rituximab: is it a BRIGHT idea?

Flinn et al . have reported the results of the long-term follow-up for the BRIGHT trial, an international study which compared the efficacy and the safety of bendamustine plus rituximab (BR) with either rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) or rituximab plus cyclophosphamide, vincristine, and prednisone (R-CVP) for treatment-naive patients with indolent non-Hodgkin lymphoma (iNHL) or mantle-cell lymphoma (MCL)

Functional dosing of mesenchymal stromal cell-derived extracellular vesicles for the prevention of acute graft-versus-host-disease

Graft-vs-host-disease (GvHD) is currently the main complication of allogeneic hematopoietic stem cell transplantation. Mortality and morbidity rates are particularly high, especially in steroid-refractory acute GvHD (aGvHD). Immune regulatory human bone marrow mesenchymal stromal cells (hMB-MSCs) represent a therapeutic approach to address this issue. Unfortunately, their effect is hardly predictable in vivo due to several variables, that is, MSC tissue origin, concentration, dose number, administration route and timing, and inflammatory status of the recipient. Interestingly, human bone marrow MSC-derived extracellular vesicles (hBM-MSC-EVs) display many of the hBM-MSC immunoregulatory properties due to their content in paracrine factors that greatly varies according to the collection method. In this study, we focused on the immunological characterization of hBM-MSC-EVs on their capability of inducing regulatory T-cells (T-regs) both in vitro and in a xenograft mouse model of aGvHD. We correlated these data with the aGvHD incidence and degree following hBM-MSC-EV intravenous administration. Thus, we first quantified the EV immunomodulation in vitro in terms of EV immunomodulatory functional unit (EV-IFU), that is, the lowest concentration of EVs leading in vitro to at least threefold increase of the T-regs compared with controls. Second, we established the EV therapeutic dose in vivo (EV-TD) corresponding to 10-fold the in vitro EV-IFU. According to this approach, we observed a significant improvement of both mouse survival and control of aGvHD onset and progression. This study confirms that EVs may represent an alternative to whole MSCs for aGvHD prevention, once the effective dose is reproducibly identified according to EV-IFU and EV-TD definition

The rs1001179 SNP and CpG methylation regulate catalase expression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (CAT) expression identifies patients with an aggressive clinical course. Elucidating mechanisms regulating CAT expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the CAT promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of CAT expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher CAT expression compared with cells bearing the CC genotype. CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with CAT levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating CAT expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher CAT levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating CAT expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of CAT, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL

Biotin-targeted Pluronic® P123/F127 mixed micelles delivering niclosamide: A repositioning strategy to treat drug-resistant lung cancer cells

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic1 P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic1 F127 was conjugated with biotin, while Pluronic1 P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P < 0.01). To go in depth into the actual therapeutic potential of NCL-loaded PMM, a cisplatin-resistant A549 lung cancer cell line (CPr-A549) was developed and its multidrug resistance tested against common chemotherapeutics. Free NCL was able to overcome chemoresistance showing cytotoxic effects in this cell line ascribable to nucleolar stress, which was associated to a significant increase of the ribosomal protein rpL3 and consequent up-regulation of p21. It is noteworthy that biotin- decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance

Continous Treatment with Ibrutinib in 100 Untreated Patients with TP53 Disrupted Chronic Lymphocytic Leukemia. A Real-Life Campus CLL Study

Continous Treatment with Ibrutinib in 100 Untreated Patients with TP53 Disrupted Chronic Lymphocytic Leukemia. A Real-Life Campus CLL Stud

Multi-component bioresponsive nanoparticles for synchronous delivery of docetaxel and TUBB3 siRNA to lung cancer cells

Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to ‘design in’ selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (β-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for βIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed “on demand” drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of β-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer

COVID-19 severity and mortality in patients with CLL: an update of the international ERIC and Campus CLL study

Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to Coronavirus disease 2019 (COVID-19) due to age, disease, and treatment-related immunosuppression. We aimed to assess risk factors of outcome and elucidate the impact of CLL-directed treatments on the course of COVID-19. We conducted a retrospective, international study, collectively including 941 patients with CLL and confirmed COVID-19. Data from the beginning of the pandemic until March 16, 2021, were collected from 91 centers. The risk factors of case fatality rate (CFR), disease severity, and overall survival (OS) were investigated. OS analysis was restricted to patients with severe COVID-19 (definition: hospitalization with need of oxygen or admission into an intensive care unit). CFR in patients with severe COVID-19 was 38.4%. OS was inferior for patients in all treatment categories compared to untreated (p < 0.001). Untreated patients had a lower risk of death (HR = 0.54, 95% CI:0.41–0.72). The risk of death was higher for older patients and those suffering from cardiac failure (HR = 1.03, 95% CI:1.02–1.04; HR = 1.79, 95% CI:1.04–3.07, respectively). Age, CLL-directed treatment, and cardiac failure were significant risk factors of OS. Untreated patients had a better chance of survival than those on treatment or recently treated