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JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells., (© 2024. The Author(s).)

Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T-cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T-cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA-4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. CTLA-4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T-cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, anti-CTLA-4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co-culture system with cytotoxic T cells. To test CTLA-4 inhibition in vivo, patient-derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA-4 blockade reduced human myeloid chimerism and led to T-cell expansion in spleen and bone marrow. Overall, these findings shed light on T-cell dysfunction in MF and suggest that CTLA-4 blockade can boost the cytotoxic T cell-mediated immune response against tumor cells., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

Transformation from chronic (CP) to blast phase (BP) in myeloproliferative neoplasm (MPN) remains poorly characterized, and no specific mutation pattern has been highlighted. BP-MPN represents an unmet need, due to its refractoriness to treatment and dismal outcome. Taking advantage of the granularity provided by single-cell sequencing (SCS), we analyzed paired samples of CP and BP in 10 patients to map clonal trajectories and interrogate target copy number variants (CNVs). Already at diagnosis, MPN present as oligoclonal diseases with varying ratio of mutated and wild-type cells, including cases where normal hematopoiesis was entirely surmised by mutated clones. BP originated from increasing clonal complexity, either on top or independent of a driver mutation, through acquisition of novel mutations as well as accumulation of clones harboring multiple mutations, that were detected at CP by SCS but were missed by bulk sequencing. There were progressive copy-number imbalances from CP to BP, that configured distinct clonal profiles and identified recurrences in genes including NF1, TET2, and BCOR, suggesting an additional level of complexity and contribution to leukemic transformation. EZH2 emerged as the gene most frequently affected by single nucleotide and CNVs, that might result in EZH2/PRC2-mediated transcriptional deregulation, as supported by combined scATAC-seq and snRNA-seq analysis of the leukemic clone in a representative case. Overall, findings provided insights into the pathogenesis of MPN-BP, identified CNVs as a hitherto poorly characterized mechanism and point to EZH2 dysregulation as target. Serial assessment of clonal dynamics might potentially allow early detection of impending disease transformation, with therapeutic implications., (© 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Background: Pemphigus is a life-threatening blistering autoimmune disease. Several forms, characterized by the presence of autoantibodies against different autoantigens, have been described. In Pemphigus Vulgaris (PV), autoantibodies target the cadherin Desmoglein 3 (DSG3), while in Pemphigus foliaceous (PF) autoantibodies target the cadherin Desmoglein 1 (DSG1). Another variant, mucocutaneous Pemphigus, is characterized by the presence of IgG against both DSG1 and DSG3. Moreover, other forms of Pemphigus characterized by the presence of autoantibodies against other autoantigens have been described. With regard to animal models, one can distinguish between passive models, where pathological IgG are transferred into neonatal mice, and active models, where B cells deriving from animals immunized against a specific autoantigen are transferred into immunodeficient mice that develop the disease. Active models recreate PV and a form of Pemphigus characterized by the presence of IgG against the cadherin Desmocollin 3 (DSC3). Further approaches allow to collect sera or B/T cells from mice immunized against a specific antigen to evaluate the mechanisms underlying the onset of the disease. Objective: To develop and characterize a new active model of Pemphigus where mice express auto antibodies against either DSG1 alone, or DSG1 and DSG3, thereby recapitulating PF and mucocutaneous Pemphigus, respectively. In addition to the existing models, with the active models reported in this work, it will be possible to recapitulate and mimic the main forms of pemphigus in adult mice, thus allowing a better understanding of the disease in the long term, including the benefit/risk ratio of new therapies. Results: The new DSG1 and the DSG1/DSG3 mixed models were developed as proposed. Immunized animals, and subsequently, animals that received splenocytes from the immunized donors produce a high concentration of circulating antibodies against the specific antigens. The severity of the disease was assessed by evaluating the PV score, evidencing that the DSG1/DSG3 mixed model exhibits the most severe symptoms among those analyzed. Alopecia, erosions, and blistering were observed in the skin of DSG1, DSG3 and DSG1/DSG3 models, while lesions in the mucosa were observed only in DSG3 and DSG1/DSG3 animals. The effectiveness of the corticosteroid Methyl-Prednisolone was evaluated in the DSG1 and DSG1/DSG3 models, that showed only partial responsiveness.

Myeloproliferative neoplasms (MPNs) are clonal disorders originated by the serial acquisition of somatic mutations in hematopoietic stem/progenitor cells. The major clinical entities are represented by polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), that are caused by driver mutations affecting JAK2 , MPL or CALR . Disease progression is related to molecular and clonal evolution. PV and ET can progress to secondary myelofibrosis (sMF) but can also evolve to secondary acute myeloid leukemia (sAML). PMF is associated with the highest frequency of leukemic transformation, which represents the main cause of death. sAML is associated with a dismal prognosis and clinical features that differ from those of de novo AML. The molecular landscape distinguishes sAML from de novo AML, since the most frequent hits involve TP53 , epigenetic regulators, spliceosome modulators or signal transduction genes. Single cell genomic studies provide novel and accurate information about clonal architecture and mutation acquisition order, allowing the reconstruction of clonal dynamics and molecular events that accompany leukemic transformation. In this review, we examine our current understanding of the genomic heterogeneity in MPNs and how it affects disease progression and leukemic transformation. We focus on molecular events elicited by somatic mutations acquisition and discuss the emerging findings coming from single cell studies.

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens.

The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.

Multicentric Castleman's Disease (MCD) is an atypical lymphoproliferative disorder, related to human herpesvirus 8 (HHV-8) infection and often associated with autoimmune diseases such as haemolytic anaemia and thrombocytopenia. Acquired haemophilia (AH) is a rare, life-threatening disease, which can occur in association with lymphoproliferative disorders, although only one case of AH in MCD has been described so far. We report the case of a human immuno deficiency virus negative 71-yr-old woman referred to our hospital for prolonged bleeding on surgical site following a lymph node biopsy. Lymph node histology revealed MCD, while the screening for the bleeding disorder showed prolonged activated partial thromboplastin time (APTT) (ratio: 1.89, normal value <1.24), low factor VIII (FVIII:C) levels (6%) with anti-factor VIII antibodies (2.3 Bethesda units), leading to a diagnosis of AH. Virological studies on plasma, lymphocyte and bronchoalveolar wash showed positivity for HHV-8 infection. Treatment with steroids (metilprednisolone 1-1.5 mg/kg/d) and cyclophosphamide (100 mg/d orally) was unsuccessful, and then antiviral therapy with cidofovir (5 mg/kg/wk) was started. A transient normalisation of APTT was seen after two administrations of cidofovir, but then coagulation parameters worsened and a large haematoma of the arm appeared. Bleeding was successfully stopped with two boluses of recombinant activated factor VII (Novoseven 90 microg/kg). Therapy with anti-CD 20 monoclonal antibody rituximab (Mabthera 375 mg/m2 once a week for 4 wk) was started, and following two administrations APTT normalised once again. Cardiological and neurological complications arose before the third dose of rituximab and the patient died shortly afterwards.

A case of vertebral artery dissection and consequent basilar artery thrombosis in a 6-year-old patient is reported. The patient was treated with subcutaneous low-molecular-weight heparin at therapeutic doses (enoxaparin, 100 IU/kg twice daily) for 3 weeks, then reduced to 2,000 IU/day for 3 more weeks. After this time a magnetic resonance angiogram was obtained that showed complete recanalization of the left basilar artery. Enoxaparin proved to be a safe and useful therapy for thrombosis accompanying vertebral artery dissection.

Treatment of severe, chronic idiopathic thrombocytopenic purpura (ITP) refractory to most usual therapies is a difficult challenge. Little information exists on the clinical use of cyclosporin A (CyA) in the treatment of ITP. This report describes long-term treatment with CyA (median, 40 months) and follow-up (median, 36.8 months) in 12 adult patients with resistant ITP. CyA used in relatively low doses (2.5-3 mg/kg of body weight per day) led to a clinical improvement in 10 patients (83.3%). Five had a complete response (41.1%), 4 a complete response to maintenance therapy (33.3%), and one a partial response (8.3%). Two patients had no response. Most patients with a response (60%) had a long-term remission (mean, 28.6 months) after discontinuation of CyA. One patient had a relapse of ITP 4 years after CyA therapy was stopped. Side effects were moderate and transient, even in patients dependent on continued CyA treatment. CyA seems to represent reasonable salvage treatment in severe, potentially life-threatening, refractory ITP.

We report on long-term treatment (13-62 months) with cyclosporin A (CyA) in eight patients with autoimmune haematological disorders, resistant to all usual therapies. Three patients had an autoimmune haemolytic anaemia (AHA); four an idiopathic thrombocytopenic purpura (ITP), and one an Evans syndrome. All patients were responsive: six achieved complete remission and two partial remission. The side-effects were moderate and transient. The majority of surviving patients remain dependent on continued drug administration. The CyA appears to be recommendable as a salvage treatment in life-threatening, resistant autoimmune haematological diseases.