Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A). The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date. Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members. Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C). In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. Figure 1 Figure 1. Disclosures Voorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Lymphoid blast transformation in an MPN with BCR-JAK2 treated with ruxolitinib: putative mechanisms of resistance

The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

Comparative characteristics of the state of the immune system in patients with coronary artery disease with stable angina pectoris and acute coronary syndrome

The aim – to assess the relationship between the state of the immune system and the development of acute coronary syndrome in patients with IHD.Materials and methods. The first group consisted of 64 patients with ST-segment elevation acute coronary syndrome, mean age 54 (49–64) years; the second group – 223 patients with coronary artery disease with stable exertional angina, FC II–III, mean age 56 (49–63) years; the third group – 47 patients with acute coronary syndrome without ST segment elevation, mean age 61 (52–65) years. The material for the immunological study was peripheral venous blood. To determine the parameters of cellular and humoral innate and adaptive immunity in blood serum and supernatants of mononuclear cells, enzyme immunoassay was used.Results and discussion. In patients with coronary artery disease with acute coronary syndrome with ST segment elevation compared with patients with coronary artery disease with stable angina pectoris, the levels of indicators of the immune status in the blood were: CRP – 9.3 (5.3–12.0) versus 4.8 (2.4–8.1) mg/L (p=0.0001), sICAM – 785 (690–830) versus 565 (406–744) ng/ml (p=0.0001), IL-10 in blood mononuclear cells – 48 (1–228) versus 194 (21–758) pg/ml (p=0.0007), circulating immune complexes – 90 (70–108) versus 76 (54–105) od. (p=0.045), lymphocytes with apoptosis (CD95) – 16 (9–27) versus 11 (8–17) % (p=0.029), spontaneous oxygen-dependent metabolism of monocytes – 16 (12–21) versus 13 (9–17) (p=0.001). The levels of indicators of the immune system in the blood in patients with coronary artery disease with acute coronary syndrome with ST segment elevation compared with patients with coronary artery disease with acute coronary syndrome without ST segment elevation were: T-helpers – 37 (32–41) versus 42 (37–48) % (p=0.0006) (R=–0.33; p=0.0005), reaction of lymphocyte blast transformation to nonspecific antigen – 38 (32–47) versus 50 (42–61) % (p=0.0004) (R=–0.37; p=0.0003).Conclusions. The development of acute coronary syndrome is directly combined with increased activity of the immune system, as evidenced by the high production of proinflammatory CRP, IL-8, sICAM with a low level of anti-inflammatory IL-10, a pronounced humoral adaptive immune response (in terms of antibodies to the myocardium and vascular tissues, CD40, circulating immune complexes) and active functional state of monocytes (according to cNCT test, functional reserve, phagocytosis) in patients with coronary artery disease with acute coronary syndrome, regardless of the position of the ST segment in comparison with patients with stable coronary artery disease. Elevated levels of antibodies to the myocardium in patients with stable coronary heart disease indicate moderate myocardial damage due to temporary ischemia in angina attacks, even with a stable course of the disease. In patients with acute coronary syndrome, high levels of antibodies to the myocardium indicate myocardial damage due to increased ischemia in plaque destabilization much earlier than the clinical manifestations of acute coronary syndrome. In acute coronary syndrome with ST-segment elevation, compared with ACS patients without ST-segment elevation, activation of neutrophils and suppression of the activity of adaptive T-cell immunity is noted (by the level of T-helpers, sCD40L, blast transformation of lymphocytes, γ-interferon in mononuclear cells, apoptosis of lymphocytes).

Clonal Megakaryocyte Dysplasia with Normal Blood Values Is a Distinct Myeloproliferative Neoplasm

<b><i>Introduction:</i></b> In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity. <b><i>Methods:</i></b> This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis. <b><i>Results:</i></b> Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41–54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. <i>JAK2</i>V617F was present in 10 subjects and <i>CALR</i> mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40–160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5–4.0) and 5.0 events (4.6–5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis. <b><i>Conclusion:</i></b> Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.

Taraxacum Officinale Alcoholic Extract Augments the Cell-Mediated Immune Response in Anti-Anthrax Vaccinated Sheep

Phytotherapy used various preparations in both human and veterinary medicine over time, due to their increased bioavailability and lower costs. Taraxacum officinale of Compositae family, well-known for its therapeutic effects, abundantly grows on Romanian pastures. The research aimed to investigate its alcoholic extract’s effects on cell-mediated immunity in anti-anthrax vaccinated, grazing sheep. Local Turcana ewes kept on pasture from spring to fall were sc vaccinated with R1190 attenuated anthrax strain, and blood was sampled before and 14 days after the vaccination. In vitro cell-mediated immunity was monitored employing phagocytosis and blast transformation tests in the presence of T. officinale alcoholic extract. The extract increased phagocytosis two weeks after the vaccination (p<0.001), while the blastogenic response also showed significant variations (p<0.05) over time. There was a positive effect of T. officinale extract on the cellular immunity, suggesting its stress alleviating effects in anti-anthrax vaccinated sheep.

Acute Myeloid Leukemia Evolving from Myeloproliferative Neoplasms: Many Sides of a Challenging Disease

The evolution to blast phase is a frequently unpredictable and almost invariably fatal event in the course of myeloproliferative neoplasms. The molecular mechanisms underlying blast transformation have not been elucidated and the specific genetic and epigenetic events governing leukemogenesis remain unclear. The result of the long-lasting dynamics, passing through progressive genetic steps, is the emergence of one or more clones often characterized by complex genetics, either at conventional karyotyping or at modern high-throughput sequencing analyses, with all clinical and prognostic correlates. The current therapeutic approaches are largely inadequate and incapable of modifying the inherent unfavorable outcome. In this perspective, the application of targeted strategies should aim to prevent the occurrence of leukemic evolution. At transformation, the crucial target of treatment should be the allocation to allogeneic transplant for eligible patients. With this in mind, novel combination treatments may provide useful bridging strategies, beyond potentially improving outcomes for patients who are not candidates for intensive approaches.

MICROBIAL CHARACTERISTICS OF POST-TRAUMATIC OSTEOMYELITIS

Objective: The one of the most important issues in traumatology is prevention and treatment of purulent-septic complications of traumatic diseases. The aim of our study was to establish correlations between osteomyelitis caused by bacterial flora and immunological factors. Methods: On the basis of a comprehensive study of bacteriological and immunological data in 100 patients with various etiologies osteomyelitis, using correlation analysis was determined: that the types of microbial complications following trauma and the date of the body's immune system depends on etiological factors. The frequency of microbes is different and depends on the localization of the injury and the surgical intervention. Results: frequency of the etiological factors in the contingent we studied, was distributed as follows: S. aureus-36,9%, S.Epidermidis-52,4, Ps. aeruginosa 27,4%, E. coli- 13,1%, Proteus- 27,4%. Associated infection (or co-infection e.g. S. aureus+S. Epidermidis, or St.Epidermidisis+Ps. Aeruginosa and etc) occurred in 22.6% of cases. A significantly high correlation coefficient was observed in patients who came to the clinics spontaneously or with delay, as well as with the early onset of the infectious process (up to two weeks) and surgical treatment. It also correlates positively with a decrease of following immunological parameters: NK, CD4+, CD8+ and CD 19+, the leucocytes phagocytic index is reduced and the blast transformation reaction of lymphocytes rate was increased. There is a significant correlation with benign outcome of treatment, which indicates that, the patient was sent for outpatient treatment (R=0, 79). Keywords: Trauma, Osteomyelitis, Microorganism, Etiological factor, Immune system data.

miR-181d/RBP2/NF-κB p65 Feedback Loop Promotes Chronic Myeloid Leukemia Blast Crisis

Background: Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease. Once it progresses into the phase of blast crisis (CML-BP), the curative effect is poor, and the fatality rate is extremely high. Therefore, it is becoming urgent to explore the molecular mechanisms of blast crisis transition and develop new therapeutic targets.Methods: The expression levels of miR-181d, RBP2 and NF-κB p65 were assessed in 42 newly diagnosed CML-CP patients and 15 CML-BP patients. Quantitative real-time PCR, Western blots, and cell proliferation assay were used to characterize the changes induced by overexpression or inhibition of miR-181d or RBP2 or p65. Luciferase reporter assay and CHIP assay was conducted to establish functional association between miR-181d, RBP2 and p65. Inhibition of miR-181d expression and its consequences in tumor growth was demonstrated in vivo models.Results: We found that the non-coding RNA miR-181d is overexpressed in CML-BP, which promotes leukemia cell proliferation. We identified histone demethylase RBP2 as directly downregulated by miR-181d and found that RBP2 inhibited p65 expression in leukemia cells by its binding to the p65 promoter and demethylating the tri/dimethylated H3K4 region in the p65 promoter locus. Conversely, p65 directly binds to miR-181d promoter and upregulates its expression; thus, forming a positive feedback loop.Conclusions: Taken together, the miR-181d/RBP2/p65 loop promotes CML blast transformation.

Immunomodulatory Activity and Phytochemical Profile of Infusions from Cleavers Herb

Extracts from aerial parts of G. aparine (cleavers) constitute a herbal remedy with monography in British Herbal Pharmacopeia. On the European market, there are several drugs and food supplements consisting of Galium extracts. In folk medicine, cleavers was used topically in Europe, Asia, and the Americas to treat skin diseases. In several remedies, cleavers is also listed as an immunomodulatory active herb influencing the defense response of the human body. The aim of this study was to investigate the immunostimulatory activity and antioxidant potential in vitro of a raw infusion of cleavers and bioactive fractions. The functional activity of lymphocytes in the reaction of the lymphocyte blast transformation (RLBT) method was used for immunomodulatory activity assays and direct scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H2O2) was chosen for the examination of antioxidant activity. It was shown that both the raw extract and fractions show significant immunostimulatory and scavenging activities. The obtained data partially justify the traditional use of cleavers as topical remedy for skin infections and for wounds.