Ibrutinib as first line therapy in chronic lymphocytic leukemia patients over 80 years old: A retrospective real-life multicenter Italian cohort.

Although chronic lymphocytic leukemia (CLL) predominantly affects the elderly, limited data exists about the outcomes of over 80-year-old patients, usually underrepresented in clinical trials. We conducted a multicenter study enrolling 79 consecutive CLL patients ≥80 years at the time of frontline therapy, all treated with ibrutinib. Nearly 48% of cases exhibited unmutated IGHV genes, 32% 17p deletion, and 39.2% TP53 mutations; 63.3% displayed a cumulative illness rating scale (CIRS) > 6. The overall response rate on ibrutinib, computed in 74/79 patients (5 patients excluded for early withdrawal), was 89.9%. After a median follow-up of 28.9 months, the median progression-free survival (PFS) and overall survival (OS) were 42.5 and 51.8 months, respectively. CIRS>6 and temporary discontinuation of ibrutinib lasting for 7-30 days were the only parameters associated with a significantly shorter PFS and were both relevant in predicting a shorter PFS compared to patients with CIRS≤6 and therapy discontinuation ≤7 days. The most common grade≥3 adverse events were infections (25.5%), neutropenia (10.1%), and anemia (2.5%). Eighteen patients (22.8%) experienced a cardiovascular event, including grade-2 atrial fibrillation (n = 9; 11%), grade-2 hypertension (n = 5; 6%), heart failure (n = 3; 3%), and acute coronary syndrome (n = 1; 1%). Mild bleeding events were observed in 27 patients (34.2%). Ibrutinib was permanently discontinued in 26 patients due to progressive disease (n = 11, including 5 Richter's syndromes), secondary malignancies (n = 6), infections (n = 3), cardiac failure (n = 3), severe bleeding (n = 2), and sudden death (n = 1). In conclusion, our analyses confirmed the overall effectiveness and favorable safety profile of the ibrutinib-single agent therapeutic approach in CLL patients ≥80 years.

Neutrophil to lymphocyte ratio in myelofibrosis patients treated with ruxolitinib may predict prognosis and rate of discontinuation.

BACKGROUND: Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloproliferative neoplasm (Ph-MPN). MF is featured by an inflammatory condition that can also drive the progression of disease. Ruxolitinib (ruxo) is the-first-in-class Jak1/2 inhibitor approved for treatment of MF, proved to reduce spleen volume and decrease symptom burden. In various malignancies neutrophil-to-lymphocyte ratio (NLR) has been indicated as predictor of progression free survival (PFS) and overall survival (OS). NLR might reflect the balance between systemic inflammation and immunity and is emerging as a prognostic biomarker in several neoplasms, including the hematological ones. METHODS: We analyzed a cohort of 140 MF patients treated with ruxo to validate baseline NLR (as a continuous variable and as a cut-off 2) as predictor of OS and of ruxo treatment discontinuation. RESULTS: We found that both baseline NLR as a continuous variable [HR 0.8 (95% CI: 0.7-0.9) (p = .006)] and NLR (<2 vs. ≥2) [HR 3.4 (95% CI: 1.6-7.0) (p = .001)] were significantly associated with OS. Censoring for patients undergone allotransplant, baseline NLR <2 was predictive of an earlier ruxo any-other-cause discontinuation [HR 3.7 (95%CI 1.7-8.3) (p < .001)]. CONCLUSIONS: NLR before starting ruxo treatment may be used as a simple and early predictor of OS and earlier ruxo discontinuation in clinical practice.

Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/ß family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

Safety and effectiveness of ruxolitinib in the real-world management of polycythemia vera patients: a collaborative retrospective study by pH-negative MPN latial group.

Ruxolitinib is approved for polycythemia vera (PV) patients after failure to previous cytoreductive therapy, based on durable results observed in phase 3 trials. We report a multicenter retrospective study demonstrating the efficacy and safety of ruxolitinib in real-life setting. Eighty-three patients were evaluated. Median follow-up was 24.5 months (IQR 14.0-29.3). At a 3-month response assessment, ruxolitinib provided significant benefit in reducing hematocrit (HCT) level (p < 0.001), phlebotomy requirement (p < 0.001), leucocytes (p = 0.044), and disease-related symptoms (p < 0.001). The exposure-adjusted rates (per 100 patient-years) of infectious complications, thromboembolic events, and secondary malignancies were 6.9, 3, and 3.7, respectively. Non-melanoma skin cancers (NMSC) were the most frequent (40%) SM type. Lymphoproliferative disorders were not detected. Five (6%) patients permanently discontinued ruxolitinib treatment and four (5%) evolved in myelofibrosis (MF), but none in acute leukemia. The rate of MF evolution per 100 patient-years of exposure was 2.8. In our experience, ruxolitinib confirmed its efficacy and safety outside of clinical trials.

E3 ubiquitin ligase PARK2, an inhibitor of melanoma cell growth, is repressed by the oncogenic ERK1/2-ELK1 transcriptional axis.

Malignant melanoma, the most aggressive form of skin cancer, is characterized by high prevalence of BRAF/NRAS mutations and hyperactivation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), mitogen-activated protein kinases (MAPK), leading to uncontrolled melanoma growth. Efficacy of current targeted therapies against mutant BRAF or MEK1/2 have been hindered by existence of innate or development of acquired resistance. Therefore, a better understanding of the mechanisms controlled by MAPK pathway driving melanogenesis will help develop new treatment approaches targeting this oncogenic cascade. Here, we identify E3 ubiquitin ligase PARK2 as a direct target of ELK1, a known transcriptional effector of MAPK signaling in melanoma cells. We show that pharmacological inhibition of BRAF-V600E or ERK1/2 in melanoma cells increases PARK2 expression. PARK2 overexpression reduces melanoma cell growth in vitro and in vivo and induces apoptosis. Conversely, its genetic silencing increases melanoma cell proliferation and reduces cell death. Further, we demonstrate that ELK1 is required by the BRAF-ERK1/2 pathway to repress PARK2 expression and promoter activity in melanoma cells. Clinically, PARK2 is highly expressed in WT BRAF and NRAS melanomas, but it is expressed at low levels in melanomas carrying BRAF/NRAS mutations. Overall, our data provide new insights into the tumor suppressive role of PARK2 in malignant melanoma and uncover a novel mechanism for the negative regulation of PARK2 via the ERK1/2-ELK1 axis. These findings suggest that reactivation of PARK2 may be a promising therapeutic approach to counteract melanoma growth.

Real-life comparison of nilotinib versus dasatinib as second-line therapy in chronic phase chronic myeloid leukemia patients.

Tyrosine kinase inhibitors (TKIs), the backbone of treatment for chronic phase chronic myeloid leukemia patients (CP-CML), have changed the long-term outcome of the disease. Nonetheless, over 20% of patients fail front-line therapy due to intolerance or resistance. A head-to-head comparison of dasatinib and nilotinib as second-line treatment outside of sponsored clinical trials has not been reported. We retrospectively analyzed 131 CP-CML patients who, after front-line imatinib failure, switched to a second-line therapy with nilotinib (59, 45%) or dasatinib (72, 55%). Median duration of second-line treatment was 33 months (range 2-100). The reason for switching therapy was resistance in 83.2% and intolerance in 16.8% of patients. The overall survival of the entire cohort at 7 years was 78.9%, while it was 72% and 85.6% for patients treated with dasatinib and nilotinib, respectively (p=0.287). With regard to efficacy after 12 months of treatment, 108 patients were evaluable for molecular response: 47% achieved a major molecular response and 18.2% a deep molecular response with dasatinib, compared to 38% and 16.2% with nilotinib (p=ns). We observed 35% of grade 3-4 adverse events, more frequently in the dasatinib group (47%) compared to the nilotinib group (22%), without affecting molecular responses. Our study suggests that, in the real-life setting, dasatinib and nilotinib used as second-line treatment in CP-CML are equally effective, with high molecular response rates and an acceptable tolerability.

Predictive factors for response and survival in elderly acute myeloid leukemia patients treated with hypomethylating agents: a real-life experience.

Predictive factors of response to hypomethylating agents (HMA) in elderly acute myeloid leukemia (AML) patients remain unclear in the real-life setting and no direct comparison between azacitidine (AZA) and decitabine (DEC) has been carried out. We retrospectively evaluated 110 AML patients treated with HMA (78 AZA, 32 DEC) as first-line therapy outside of clinical trials. Median age was 75 years (range 58-87). The median overall survival (OS) of the entire cohort was 8.0 months (95% CI 6.1-10), without significant differences among the subgroups: AZA 8.8 months vs DEC 6.3 months (p = 0.291). HMA treatment yielded an overall response rate (ORR) of 40% (AZA 37% vs DEC 47%, p = 0.237). A stable disease (SD) after 4 HMA cycles was not associated with a worse survival outcome compared with an early optimal response. Factors independently associated with a better OS were transfusion independence during treatment (p = 0.049), achievement of an optimal response to treatment (p < 0.001), and a baseline hemoglobin level ≥ 9.25 (p = 0.018). A bone marrow (BM) blast count ≥ 30% (p < 0.001) and a therapy-related AML (p = 0.008) remain poor survival predictors. Of the available biologic features, an adverse risk category according to the ELN classification was significantly associated with a shorter survival over the intermediate risk category (p = 0.034). Disease progression remains the primary cause of death. Infectious complications were more severe (p = 0.036) and occurred earlier (p = 0.006) in the DEC group compared with that of the AZA group. In conclusion, clinical prognostic factors associated to response and survival have been identified without significant associations concerning overall outcomes between the two HMAs.

Switch from branded to generic imatinib: impact on molecular responses and safety in chronic-phase chronic myeloid leukemia patients.

Since July 2017, different generic imatinib formulations have been introduced in Italy for the treatment of patients with chronic myeloid leukemia (CML). We analyzed 168 chronic phase CML patients treated with branded imatinib for a median of 12 years (range 1-16) at a single institution who switched to a single generic formulation in order to assess the safety and impact on molecular response. The Sokal risk was low/intermediate/high in 63%, 33%, and 4% of patients, respectively. The median duration of generic imatinib treatment was 19 months (range 4-22). Twenty-seven percent of patients were in MMR and 73% were in deep molecular responses (MR4-4.5) at the time of the switch. After 12 months of treatment with generic imatinib, 140 patients were evaluable for response: 23.6% and 76.4% were respectively in MMR and in deep molecular response. When the degree of response was compared with the best molecular response observed with branded imatinib, it was found that 84% of patients maintained the response previously achieved, 6% improved it, and 10% of patients had a molecular fluctuation from the previous deep molecular response to MMR. Only 1 patient lost the MMR and no patient switched to another TKI for inefficacy. In terms of safety, 20% of patients reported new or worsening side effects, but only 2 patients returned to branded imatinib for toxicity. Our data show that the switch to generic imatinib in patients who have been previously treated with branded imatinib appears to maintain efficacy, although a proportion of patients experience new or worsening side effects.

ATP6V1A is required for synaptic rearrangements and plasticity in murine hippocampal neurons.

AIM: Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. METHODS: Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. RESULTS: Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH-regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. CONCLUSIONS: These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties.

Validation of imatinib therapy failure score (IMTF) in chronic phase chronic myeloid leukemia in real life practice.

The outcome of chronic myeloid leukemia (CML) patients improved in the last decade. Clinical prognostic scoring systems aim to provide information about survival in the long-term, without determining from baseline the subset of patients who require a strictly monitoring because at increased risk of failure. Imatinib, the first-generation tyrosine kinase inhibitor (TKI), is still widely used as frontline treatment: recently, the imatinib therapy failure (IMTF) score was proposed to identify the failure free survival. Aim of our study was to validate this index in a large cohort of patients treated with imatinib.

Complication of Gastric Cancer Surgery: A Single Centre Experience.

BACKGROUND/AIM: Gastric cancer surgery is still characterised by high morbidity and mortality. However, in 2018 an online platform, GASTRODATA has been proposed in Europe to standardize the recording of gastric surgery complications. The aim of the study was to present a single center experience regarding incidence and grading of acute postoperative complications in a population of patients treated surgically for gastric cancer on the basis of the gastrodata online platform. PATIENTS AND METHODS: The present study was a single center, observational, retrospective trial held in the General Surgery Unit of the Sant'Andrea Hospital of Rome. The study included 181 consecutive patients who underwent gastric surgical resection for cancer from May 2004 to December 2020 with curative R0 purpose. RESULTS: Thirty-three percent of patients reported at least one complication, while seventeen percent of the whole population reported a complication classified as at least grade 3 on the Clavien Dindo Classification. The most frequent complications were disorders of the respiratory system (13.3%), followed by bleeding (7.6%) and wound infections (6.2%). Deaths accounted for 3.7% of the population. CONCLUSION: A list of defined complications of gastrectomy, if systematically adopted in the Literature, could lead to a reduction in the wide variation of proposals for treatment and assessment. Objectively evaluating the impact of complications on outcomes can lead to quality improvement project proposals.

A micropatterned thermoplasmonic substrate for neuromodulation of in vitro neuronal networks.

Understanding how the spatial organization of a neural network affects its activity represents a leading issue in neuroscience. Thanks to their accessibility and easy handling, in vitro studies remain an essential tool to investigate the relationship between the structure and function of a neuronal network. Among all the patterning techniques, ink-jet printing acquired great interest thanks to its direct-write approach, which allows the patterned substrate realization without mold, leading to a considerable saving of both cost and time. However, the inks commonly used give the possibility to control only the structure of a neuronal network, leaving aside the functional aspect. In this work, we synthesize a photosensitive ink combining the rheological and bioadhesive properties of chitosan with the plasmonic properties of gold nanorods, obtaining an ink able to control both the spatial organization of a two-dimensional neuronal network and its activity through photothermal effect. After the ink characterization, we demonstrate that it is possible to print, with high precision, different geometries on a microelectrode array. In this way, it is possible obtaining a patterned device to control the structure of a neuronal network, to record its activity and to modulate it via photothermal effect. Finally, to our knowledge, we report the first evidence of photothermal inhibition of human neurons activity. STATEMENT OF SIGNIFICANCE: Patterned cell cultures remain the most efficient and simple tool for linking structural and functional studies, especially in the neuronal field. Ink-jet printing is the technique with which it is possible to realize patterned structures in the fastest, simple, versatile and low-cost way. However, the inks currently used permit the control only of the neuronal network structure but do not allow the control-modulation of the network activity. In this study, we realize and characterize a photosensitive bioink with which it is possible to drive both the structure and the activity of a neuronal network. Moreover, we report the first evidence of activity inhibition by the photothermal effect on human neurons as far as we know.

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

Case Report: Severe cutaneous adverse event associated with checkpoint inhibition in the setting of CAR T-cell therapy: beyond CRS.

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy actually represents the standard of care for multiple relapsed or refractory primary mediastinal B-cell lymphoma (r/r PMBCL). Checkpoint inhibitors, such as pembrolizumab, appear to be a safe and effective treatment strategy for patients who are ineligible for or resistant to autologous stem cell transplantation. Although preclinical studies suggested that checkpoint inhibitors may enhance the vitality and anti-tumor activity of CAR T cells, there are no substantial/robust clinical data about the immune-mediated toxicity of their association. We describe a case of a severe cutaneous adverse event arising immediately after Cytokine Release Syndrome (CRS) on day +6 from CAR T cells infusion in a young r/r PMBCL patient who previously received pembrolizumab. These skin lesions were interpreted as an immune mediated adverse event, considering their prompt improvement and fully recovering achieved with the addition of immunoglobulin infusion to systemic steroid therapy. This case of life-threatening cutaneous adverse event calls for further investigations about off-target immune-related adverse events deriving from the combination of CAR T cell therapy and checkpoint inhibition, whose synergic therapeutic effect is promising.