Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.

The Value of Nasal and Oral Clinical Examination in Febrile Neutropenic Patients for Initiating Antifungal Therapy as a Preemptive Method.

BACKGROUND: Invasive fungal infections (IFIs) are complications that lead to mortality and morbidity in hematologic malignancies. The time of starting antifungal therapy is vital. Preemptive antifungal therapy has appeared recently as a new policy for the management of IFIs based on noninvasive ways in neutropenic patients. METHODS: We enrolled leukemia patients with neutropenia after chemotherapy in Imam Khomeini Hospital Complex, Tehran, Iran. Patients who entered the neutropenic phase were divided into two categories (empirical and preemptive) for receiving antifungal agents. The patients were clinically examined in the preemptive group every day to find IFIs. As soon as clinical evidence of IFIs was observed, antifungal was prescribed. The empirical group patients received antifungals based on the ward protocol. Based on the data in each group, the diagnostic and therapeutic results of cases are followed-up to 3 months. To compare percentages between the two groups, the chi-squared test was used. And to compare two means between the two groups, the independent t-test was used. All the statistical analyses were done in the Statistical Package for the Social Sciences (SPSS) version 24 software (IBM Corporation, Armonk, New York, USA). RESULTS: We assessed 132 leukemic patients with inclusion and exclusion criteria. Eventually, 80 patients were enrolled. The mean age was 35.52 years. Demographics data and distribution of leukemia type show no significant differences between the two groups. Despite a higher percentage of IFIs discovered in the preemptive group than the empirical group (25 vs. 18.75%, respectively), but data show no significant differences. The average days of IFIs diagnosis since the beginning of neutropenia in the empirical group were 9.5 days while in the preemptive group, the average days were 5.4 days (p < 0.05). Totally, there were 15 patients with a proven IFI in each group (40% in the empirical group and 60% in the preemptive group). Results significantly show an increase in surgical sinus debridement in the empirical groups (83.3%) vs. the preemptive groups (55.5%), (p < 0.05). The mortality rate differed significantly among the two groups; it was 7.5% in the preemptive group and 25% in the empirical group (p < 0.05). CONCLUSION: Daily oral and nasal cavities examination to find the symptoms of IFIs and then start preemptive antifungal agents may be able to lead to accurate diagnosis, earlier treatment, and decreasing sinus surgery debridement in leukemia patients with neutropenia.

Safety and efficacy of granulocyte-colony-stimulating factor administration following autologous intramuscular implantation of bone marrow mononuclear cells: a randomized controlled trial in patients with advanced lower limb ischemia.

BACKGROUND AIMS: The aim was to investigate the therapeutic effect of granulocyte-colony-stimulating factor (G-CSF) administration following implantation of autologous bone marrow mononuclear cells (BM MNC) for patients with lower limb ischemia. METHODS: The design was a randomized controlled trial. Fifteen patients with severe chronic limb ischemia were treated with autologous BM MNC [without G-CSF (MNC-G-CSF) or combined with G-CSF administration for 5 days following transplantation (MNC+G-CSF)]. RESULTS: All clinical parameters, including ankle brachial index, visual analog scale and pain-free walking distance, showed a mean improvement from baseline, which was measured at 4 and 24 weeks after transplantation in both groups. However, in three (20%) patients, the clinical course did not improve and limb salvage was not achieved. No significant difference was observed among the patients treated in the MNC-G-CSF and MNC+G-CSF groups. No severe adverse reactions were reported during the study period. No relationship was observed between both the numbers of viable MNC or CD34+ cells and the clinical outcome. CONCLUSIONS: Autologous transplantation of BM MNC into ischemic lower limbs is safe, feasible and efficient for patients with severe peripheral artery disease. However, the administration of G-CSF following cell transplantation does not improve the effect of BM MNC implantation and therefore would not have any beneficial value in clinical applications of such cases.

Inhibition of tumor growth by mouse ROR1 specific antibody in a syngeneic mouse tumor model.

INTRODUCTION: Immunotherapy with tumor-associated antigens (TAAs) is a potentially powerful approach to eradicate tumor cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) plays a crucial role for survival of tumor cells and is overexpressed in various malignancies. In the present study, we developed a syngeneic mouse tumor model to assess anti-tumor effect of mouse ROR1 specific polyclonal antibody (pAb) in vivo. MATERIALS AND METHODS: Mouse ROR1 specific antibody was produced in rabbit using recombinant ROR1 protein. Tow mouse tumor cell lines, (4T1 and CT26), were transfected with full length mouse ROR1 construct and stable clones were selected and characterized by immunocytochemistry, Western blot and flow cytometry. In vitro and in vivo anti-tumor activities of anti-ROR1 antibody were assessed by XTT and syngeneic BALB/c mouse model, respectively. RESULTS: We successfully established two mouse ROR1-overexpressing tumor cell lines. The in vitro results indicate that the ROR1pAb did not significantly inhibit growth of ROR1+ cell lines. One of these cell lines (CT26-ROR1) was implanted in syngeneic BALB/c mice to assess anti-ROR1 tumor inhibitory activity in vivo. The tumor size was significantly reduced in mice treated with ROR1 specific pAb. CONCLUSION: Our results demonstrated for the first time tumor inhibitory effect of mouse ROR1 specific antibody in a syngeneic mouse tumor model. This model is a promising tool for preclinical assessment of ROR1 therapeutics and investigation of the underling molecular mechanisms.

Expression of the testis-specific gene, TSGA10, in Iranian patients with acute lymphoblastic leukemia (ALL).

Testis-specific gene antigen (TSGA10) is expressed in fetus, testis and frequently in human solid cancers and acute leukemias, making it a candidate for immunotherapy and for detection of minimal residual disease (MRD). This gene is considered as a member of cancer-testis (CT) genes. We previously demonstrated TSGA10 expression during spermatogenesis. There is also evidence for potential TSGA10 involvement in cell proliferation. TSGA10 expression has been observed in a wide spectrum of cancers but not in hematopoietic neoplasm. Here we demonstrated expression of TSGA10 by semi-quantitative RT-PCR in 44 (84.6%) out of 52 bone marrow samples and all peripheral blood samples from patients with acute lymphoblastic leukemia (ALL). Twenty-seven (52%) cases had high level of gene expression and 16 (30.7%) cases had a lower expression level of the gene in the patients bone marrow. Presence of TSGA10 expression in ALL may open a window to functional study of mitotic checkpoint proteins in leukemia. RT-PCR of TSGA10 may help in detection of residual clonal cells leading to early diagnosis and better prognostic qualification of the disease.

The quality of life of hematological malignancy patients with major depressive disorder or subsyndromal depression.

OBJECTIVE: The purpose of the present study was to compare the quality of life of hematological malignancy patients with major depressive disorder or subsyndromal depression. METHOD: Sample consisted of 93 hematological malignancy patients recruited from oncology ward of Valieasr hospital for Imam Khomeini complex hospital at Tehran through purposeful sampling. Participants were divided into three groups through diagnostic interview based on DSM-IV-TR criteria and the Beck Depression Inventory-2 (BDI-II): Major depressive disorder (MDD) (n = 41; 44.1%); subsyndromal depression (SSD) (n = 23; 24.7%), and without depression (WD) (n = 29; 31.2%). Participants completed the short-form health survey (SF-36) as a measure of the quality of life. We carried out an analysis of covariance to examine the collected data. RESULTS: Findings showed that there was not a significant difference between patients with MDD and SSD based on measure of quality of life. But patients with MDD and SSD showed significantly worse quality of life than patients with WD. This finding highlights the clinical importance of subsyndromal depressive symptoms and casts doubt on the clinical utility of separation between MDD and subsyndromal depression in terms of important clinical outcomes.

Acquired hemophilia A: clinical features, surgery and treatment of 34 cases, and experience of using recombinant factor VIIa.

Acquired hemophilia A is rare, but life-threatening disorder caused by autoantibody against factor VIII. As it is useful to gather more data on epidemiology, clinical pictures and therapy of it, we evaluated relevant medical findings in 34 acquired hemophiliacs from Dec 1999 to Dec 2007. Eight patients (23.5%) had low titers (<10 Bethesda Unit BU) and 26 patients (76.5%) had high titers of inhibitors (>10 BU). The mean of inhibitors was 548.38 +/- 359.27 SD BU. The most common hemorrhagic symptoms were hematoma 21 (33.33%), ecchymosis 16 (25.39%), hemarthrosis 8 (12.69%), hematuria 6 (9.52%), menorrhagia 4 (6.34%), compartment syndrome 3 episodes (4.76%). The eliminator therapies were recruited according to titers of inhibitor and types of bleeding and it's results were 27 efficient treatments (79.4%), 5 partial efficient treatment (14.7%) and two treatments inefficient (5.9%). Elimination therapy using steroid alone or with combination can terminate complete remission in most cases.