Accessory spleens recapitulate the immune microenvironment of the main spleen in immune thrombocytopenia.

Accessory spleens (AcS) may play a relevant role in immune thrombocytopenia (ITP) and possibly contribute to ITP relapse following splenectomy. Little is known about the immune microenvironment of AcS in ITP. To address this issue, we compared the histological features of eight matched AcS and main spleen (MS) samples, obtained from adult patients with primary ITP. AcS and MS had overlapping immune architectural features and lymphoid composition, suggesting that similar immunologic events occur in AcS and MS of ITP. These findings may have implications for the potential mechanisms of AcS-mediated ITP relapse. Further studies are needed to confirm these results and to dissect spleen immunity in ITP.

Usefulness of methotrexate in relapsing idiopathic retroperitoneal fibrosis.

OBJECTIVES: Glucocorticoids are the mainstay for treatment of retroperitoneal fibrosis (RPF), a disease characterised by a periaortic proliferation of fibroinflammatory tissue frequently causing urinary obstruction. The therapeutic approach to patients unsuitable for steroid therapy and to relapsing cases is still undefined. METHODS: In this retrospective single-centre study we evaluated 15 patients with RPF who received second-line therapy with methotrexate (MTX) between January 2011 to December 2019. RESULTS: Fourteen out of 15 patients (93%) showed response to MTX. Two patients experienced relapse: one patient when on MTX therapy (28 months), the other, 58 months after MTX was interrupted. Liver toxicity grade 2 was documented in 2 patients and resolved with temporary dosage reduction. One patient stopped MTX autonomously because of nausea. No severe infections were recorded. CONCLUSIONS: In selected patients with RPF who are intolerant or refractory to steroid single therapy, MTX may be considered as useful and safe second-line treatment.

Subcutaneous Gammanorm® by pump or rapid push infusion: Impact of the device on quality of life in adult patients with primary immunodeficiencies.

Many patients with immunodeficiencies require lifelong immunoglobulin replacement therapy (IgRT). In a multicenter, randomized, open-label, crossover, non-inferiority 3-month-trial, we compared the impact of the subcutaneous immunoglobulin Gammanorm® administered via pump or syringe (rapid push). Primary endpoint was the life quality index (LQI), secondary endpoints were QoL (SF36v2), satisfaction (TSQM-11), disease and treatment burden (PRISM), incidence of infections and adverse events (AE), treatment costs, and IgG levels. 28/30 patients completed the study. Most of the endpoints were comparable. Drug administrations with rapid push were more frequent, but reduced total time expenditure and some costs. Of the TSQM-11/LQI/SF36 components only "treatment interference with daily activities" was superior with pump and two QoL domains with rapid push. Both delivery devices showed favorable safety. Rapid push was preferred by 34.5% of patients. It proved to be an efficacious and cost-effective alternative to pumps adding to patient choice and increasing flexibility during long-term IgRT.

Thrombopoietin receptor agonists increase splenic regulatory T-cell numbers in immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RA) are a valid therapy for immune thrombocytopenia (ITP), due to megakaryocyte stimulation and (poorly characterised) immune-modulatory effects. The spleen is pivotal in the pathogenesis of ITP, yet little is known on its immune microenvironment and on effects of TPO-RA on this organ. To address these topics, we analysed 35 spleens removed for primary refractory ITP. Pre-splenectomy TPO-RA administration correlated with increased splenic regulatory T cells (Tregs), type 2 T-helper cells and histiocyte density and with reduced red pulp sinusoids. Surgical outcome was not associated with TPO-RA administration, other pre-splenectomy therapies and/or Treg density. In conclusion, TPO-RA affect the splenic microenvironment, but this has no impact on splenectomy outcome.

Histology of the spleen in immune thrombocytopenia: clinical-pathological characterization and prognostic implications.

OBJECTIVE: Immune thrombocytopenia (ITP) is an acquired disorder, characterized by immune-mediated platelet destruction. The spleen plays a key pathogenic role in ITP and splenectomy is a valuable second-line therapy for this disease. Little is known on ITP spleen histology and response to splenectomy is unpredictable. This study aims to characterize ITP spleen histology and assess possible predictors of splenectomy outcome. METHODS: A series of 23 ITP spleens were retrospectively assessed for the following histological parameters: density of lymphoid follicles (LFs), marginal zones (MZs), T helper and cytotoxic T cells; presence of reactive germinal centers (GCs); width of perivascular T cell sheaths; and red pulp features. Clinical and histological data were matched with postsplenectomy platelet counts to assess their prognostic relevance. RESULTS: Three histological patterns were documented: a hyperplastic white pulp pattern, a non-activated white pulp pattern (lacking GCs), and a white pulp-depleted pattern. Poor surgical responses were associated with presplenectomy high-dose steroid administration, autoimmune comorbidities and low T follicular helper cell density. The combination of such parameters stratified patients into different splenectomy response groups. The removal of accessory spleens was also associated with better outcome. CONCLUSION: ITP spleens are histologically heterogeneous and clinical-pathological parameters may help predict the splenectomy outcome.

A novel RUNX1 mutation with ANKRD26 dysregulation is related to thrombocytopenia in a sporadic form of myelodysplastic syndrome.

Aging is associated with a higher risk of developing malignant diseases, including myelodysplastic syndromes, clonal disorders characterised by chronic cytopenias (anaemia, neutropenia and thrombocytopenia) and abnormal cellular maturation. Myelodysplastic syndromes arising in older subjects are influenced by combinations of acquired somatic genetic lesions driving evolution from clonal haematopoiesis to myelodysplastic syndromes and from myelodysplastic syndromes to acute leukaemia. A different pattern of mutations has been identified in a small subset of myelodysplastic syndromes arising in young patients with familial syndromes. In particular, dysregulation of ANKRD26, RUNX1 and ETV6 genes plays a role in familial thrombocytopenia with predisposition to myelodysplastic syndromes and acute leukaemia. Whether these genes affect thrombopoiesis in sporadic myelodysplastic syndrome with thrombocytopenia is still undefined. Thirty-one myelodysplastic syndromes subjects and 27 controls subjects were investigated. Genomic DNA was used for mutation screening (ETV6, RUNX1, 5'UTR ANKRD26 genes). Functional studies were performed in the MEG-01-akaryoblastic cell line. We found four novel variants of RUNX1 gene, all in elderly myelodysplastic syndromes subjects with thrombocytopenia. Functional studies of the variant p.Pro103Arg showed no changes in RUNX1 expression, but the variant was associated with deregulated high transcriptional activity of ANKRD26 in MEG-01 cells. RUNX1 variant p.Pro103Arg was also associated with increased viability and reduced apoptosis of MEG-01, as well as impaired platelet production. Our findings are consistent with dysregulation of ANKRD26 in RUNX1 haploinsufficiency. Lack of repression of ANKRD26 expression may contribute to thrombocytopenia of subjects with sporadic myelodysplastic syndromes.

Coronary microvascular dysfunction due to essential thrombocythemia and policythemia vera: the missing piece in the puzzle of their increased cardiovascular risk?

Myeloproliferative neoplasms are most commonly associated with venous thrombosis. Up to 60% of patients experience a thrombotic event in their lifetimes, including stroke or myocardial infarction. It is unclear whether pathogenetic factors linking essential thrombocythemia (ET) and polycythemia vera (PV) to thrombotic complications do play a role in the risk of coronary artery disease (CAD). We aimed to assess coronary flow reserve (CFR) as a marker of coronary microvascular function in asymptomatic patients with ET and PV. Fifty-two patients with ET (M/F 13/39, age 61 ± 7 years) and 22 patients with PV (M/F 13/9, age 60.4 ± 13 years) without clinical evidence of heart disease, and 50 controls matched for age and gender were studied. None had CAD. All control subjects were asymptomatic with no history of heart disease. CFR in the left anterior descending coronary artery was detected by transthoracic Doppler echocardiography, at rest, and during adenosine infusion. In patients with ET and PV, CFR was lower than in controls (2.9 ± 0.94 and 2.2 ± 0.7 vs. 3.8 ± 0.7, P < 0.004 and P < 0.0001 respectively). The prevalence of CFR ≤ 2.5 was higher in patients with ET (20 cases, 38.5%) and PV (15 cases, 68.2%) compared with controls (4.1%) (P < 0.0001). Severe CFR (CFR < 2) impairment was found in eight patients with ET (15.4%), in nine patients with PV (40.9%), and in none of control subjects. The mutation of JAK2 gene was associated with abnormal CFR. Asymptomatic patients with ET and PV have coronary microvascular dysfunction in the absence of clinical conditions suggesting CAD.

Comparison of three different immunoassays in the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is caused by platelet activating antibodies that recognize platelet factor 4/heparin (PF4/H) complexes. Laboratory testing plays a key role in the diagnosis of HIT. As functional assays are unfeasible for most clinical laboratories, antigen binding assays are commonly used in routine testing. However, their low specificity leads to overdiagnosis of HIT. Therefore, it is advisable to improve screening tests in this setting. METHODS: Blood samples from 114 patients in whom HIT was suspected were investigated using a chemiluminescence test (HemosIL® AcuStar HIT-IgG), a PF4/H IgG enzyme immunoassay (Lifecodes PF4 IgG), an IgG-specific lateral flow immunoassay heparin-induced thrombocytopenia (LFI-HIT, STic Expert® HIT) and the heparin-induced platelet aggregation (HIPA) test. RESULTS: Twenty-nine (25.4%) out of 114 subjects with suspected HIT had a positive HIPA test. None of patients with a 4Ts score <4 were positive at HIPA. HemosIL® AcuStar HIT-IgG showed the best performance in term of sensitivity and specificity when used as single test. Receiver operating characteristic (ROC) analysis showed optimization of sensitivity and specificity using a cut-off of 1.13 U/mL (0.95 and 0.98, respectively). As an alternative approach, a strategy based on screening samples by STic Expert® HIT and then retesting positive results by Lifecodes PF4 IgG (cut-off 1 OD) or HemosIL® AcuStar HIT-IgG (cut-off 1.3 U/mL) showed a performance compared to a single test approach by HemosIL® AcuStar HIT-IgG. CONCLUSIONS: The HemosIL® AcuStar HIT or a combinatorial approach with the STic Expert® HIT and the PF4/H IgG enzyme immunoassay provide an accurate diagnosis of immune HIT.

Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy.

The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory M1-like phenotype and in facilitating CD4(+) and CD8(+) T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8(+) T-cell-dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.

MYH9-related disease: a novel prognostic model to predict the clinical evolution of the disease based on genotype-phenotype correlations.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD.

Large B-cell lymphoma with IRF4 rearrangement: a multi-centric study with focus on potential misleading phenotypes.

Large B-cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a rare lymphoid neoplasm, usually occurring in the pediatric/young-adult age. Despite this, subsets of cases occur in elderly patients and express CD5, possibly entering the differential diagnosis with adult aggressive lymphomas, such as blastoid/pleomorphic mantle cell lymphoma (MCL-B/P). To better characterize the clinical-pathological features and differential diagnosis of LBCL-IRF4, we conducted a multi-centric study on 12 cases, focusing on CD5, Cyclin D1, and SOX11 expression. While most cases had typical presentation, adult-to-elderly age at diagnosis and unusual anatomic locations were reported in 3/12 (25.0%) and 2/12 (16.7%) patients, respectively. Histologically, CD5 was positive in 4/12 (33.3%) cases, Cyclin D1 was invariably negative, and SOX11 was weakly/partially expressed in 1/12 (8.3%) case. In conclusion, LBCL-IRF4 can have unconventional clinical presentations that may challenge its recognition. Although CD5 is frequently expressed, negativity for Cyclin D1 and SOX11 contributes to the differential diagnosis with MCL-B/P.

When Waldenström macroglobulinemia hits the kidney: Description of a case series and management of a "rare in rare" scenario.

BACKGROUND: Renal injury related to Waldenström macroglobulinemia (WM) occurs in approximately 3% of patients. Kidney biopsy is crucial to discriminate between distinct histopathological entities such as glomerular (amyloidotic and non-amyloidotic), tubulo-interstitial and non-paraprotein mediated renal damage. In this context, disease characterization, management, relationship between renal, and hematological response have been poorly explored. We collected clinical, genetic and laboratory data of seven cases of biopsy-proven renal involvement by WM managed at our academic center and focused on three cases we judged paradigmatic discussing their histopathological patterns, clinical features, and therapeutic options. CASE: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. In our series AL Amyloidosis (n = 3/7) and tubulo-interstitial infiltration by lymphoma cells (n = 3/7) were the two more represented entities. BTKi did not seem to improve renal function (Case 1), while bortezomib-based regimens demonstrated a beneficial activity on the hematological and organ response, even when used as second-line therapy after chemoimmunotherapy (Case 3) and also with coexistence of anti-MAG neuropathy (Case 2). In case of poor response to bortezomib, standard chemoimmunotherapy (CIT), such as rituximab-bendamustine, represents an effective option (Case 1, 6, and 7). In our series, CIT generates durable responses more frequently in cases with amyloidogenic renal damage (Case 1, 5, and 7). CONCLUSION: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. Studies with higher numerosity are needed to better clarify the pathological and clinical features of renal involvement during WM and to determine the potential benefit of different therapeutic regimens according to the histopathological subtypes.

Enhanced and aberrant T cell trafficking following total body irradiation: a gateway to graft-versus-host disease?

Pre-transplant conditioning regimens play a major role in triggering graft-versus-host disease (GVHD). This study investigated the effect of irradiation on donor T cell trafficking to lymphoid and non-lymphoid tissues by comparing the migration of carboxy-fluorescein diacetate succinimidyl ester-labelled, naïve donor T lymphocytes in vivo in irradiated and non-irradiated syngeneic mice recipients. Recruitment of adoptively transferred naïve T cells to secondary lymphoid organs was increased in irradiated mice and naïve T cells also aberrantly localized to non-lymphoid tissues. Irradiation also induced aberrant effector memory T cell migration into lymph nodes and their localization to homing-privileged non-lymphoid sites, such as the gut. The presence of a minor histocompatibility mismatch further enhanced the aberrant accumulation of T cells in both lymphoid and non-lymphoid tissue, whilst their migratory pattern was not modified as compared to fully matched irradiated recipients. These effects correlated with decreased permeability of, and the secretion of chemotactic factors by the endothelium. Our findings are consistent with the possibility that excessive, dysregulated extravasation of T cells induced by irradiation promotes the development of GVHD.

Salvage chemoimmunotherapy with rituximab, ifosfamide and etoposide (R-IE regimen) in patients with primary CNS lymphoma relapsed or refractory to high-dose methotrexate-based chemotherapy.

Despite a high proportion of patients with primary CNS lymphoma (PCNSL) experiences failure after/during first-line treatment, a few studies focused on salvage therapy are available, often with disappointing results. Herein, we report feasibility and activity of a combination of rituximab, ifosfamide and etoposide (R-IE regimen) in a multicentre series of patients with PCNSL relapsed or refractory to high-dose methotrexate-based chemotherapy. We considered consecutive HIV-negative patients ≤75 years old with failed PCNSL treated with R-IE regimen (rituximab 375 mg/m(2) , day 0; ifosfamide 2 g/m(2) /day, days1-3; etoposide 250 mg/m(2) , day 1; four courses). Twenty-two patients (median age 60 years; range 39-72; male/female ratio: 1:4) received R-IE as second-line (n = 18) or third-line (n = 4) treatment. Eleven patients had refractory PCNSL, and 11 had relapsing disease. Twelve patients had been previously irradiated. Sixty (68%) of the 88 planned courses were actually delivered; only one patient interrupted R-IE because of toxicity. Grade 4 hematological toxicity was manageable; a single case of grade 4 non-hematological toxicity (transient hepatotoxicity) was recorded. Response was complete in six patients and partial in three (overall response rate = 41%; 95%CI: 21-61%). Seven patients were successfully referred to autologous peripheral blood stem cell collection; four responders were consolidated with high-dose chemotherapy supported by autologous stem cell transplant. At a median follow-up of 24 months, eight responders did not experience relapse, two of them died of neurological impairment while in remission. Six patients are alive, with a 2-year survival after relapse of 25 ± 9%. We concluded that R-IE is a feasible and active combination for patients with relapsed/refractory PCNSL. This regimen allows stem cell collection and successful consolidation with high-dose chemotherapy and autologous transplant.

Tixagevimab/Cilgavimab as pre-exposure prophylaxis against SARS-CoV-2 in patients with hematological malignancies.

The approved combination of Tixagevimab/Cilgavimab has been shown to decrease the rate of symptomatic SARS-CoV-2 infection in patients at increased risk of inadequate response to vaccination. However, Tixagevimab/Cilgavimab was tested in a few studies that included patients with hematological malignancies, even if this population has shown an increased risk of unfavorable outcomes following infection (with high rates of hospitalization, intensive care unit admission, and mortality) and poor significant immunization following vaccines. We performed a real-life prospective cohort study to evaluate the rate of SARS-CoV-2 infection following pre-exposure prophylaxis with Tixagevimab/Cilgavimab in anti-spike seronegative patients compared to a cohort of seropositive patients who were observed or received a fourth vaccine dose. We recruited 103 patients with a mean age of 67 years: 35 (34%) received Tixagevimab/Cilgavimab and were followed from March 17, 2022, until November 15, 2022. After a median follow-up of 4.24 months, the 3-month cumulative incidence of infection was 20% versus 12% in the Tixagevimab/Cilgavimab and observation/vaccine groups respectively (HR 1.57; 95% CI: 0.65-3.56; p = 0.34). In this study, we report our experience with Tixagevimab/Cilgavimab and a tailored approach to SARS-CoV-2 infection prevention in patients with hematological malignancies during the SARS-CoV-2 omicron surge.

CK2ß Regulates Hematopoietic Stem Cell Biology and Erythropoiesis.

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the ß regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2ß knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2ß were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.

Myocardial infarction in two cousins heterozygous for ASN41HIS autosomal dominant variant of Bernard-Soulier syndrome.

Bernard-Soulier Syndrome is characterized by thrombocytopenia with large platelets and defective aggregation to ristocetin. The bleeding tendency is variable but may be severe. The syndrome is due to genetic defects of the GPIb-V-IX complex and it has been maintained to be protective from thrombotic events. Here we present the first two cases of documented M.I. in two cousins, heterozygous for the Arg41His mutation which is responsible for a dominant form of Bernard-Soulier Syndrome. In one of the two patients an aneurysm of the aorta was also present. The patients had a mild bleeding tendency which was severely aggravated by treatment with antiplatelet drugs. These clinical observations are in contrast with experimental studies which demonstrate that Bernard-Soulier-like strains of mice show a decreased thrombus generation in several experimental settings.