Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective: Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods: We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors' and ALL/NHL patients' peripheral blood mononuclear cells. Results: We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion: This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.

A Multi-Center Study on the Efficacy of Eltrombopag in Management of Refractory Chronic Immune Thrombocytopenia: A Real-Life Experience

Objective: The aim of the present study was to evaluate the efficacy and safety of eltrombopag, an oral thrombopoietin receptor agonist, in patients with chronic immune thrombocytopenia (ITP). Materials and Methods: A total of 285 chronic ITP patients (187 women, 65.6%; 98 men, 34.4%) followed in 55 centers were enrolled in this retrospective cohort. Response to treatment was assessed according to platelet count (/mm3) and defined as complete (platelet count of >100,000/mm3), partial (30,000-100,000/mm3 or doubling of platelet count after treatment), or unresponsive (<30,000/mm3). Clinical findings, descriptive features, response to treatment, and side effects were recorded. Correlations between descriptive, clinical, and hematological parameters were analyzed. Results: The median age at diagnosis was 43.9±20.6 (range: 3-95) years and the duration of follow-up was 18.0±6.4 (range: 6-28.2) months. Overall response rate was 86.7% (n=247). Complete and partial responses were observed in 182 (63.8%) and 65 (22.8%) patients, respectively. Thirty-eight patients (13.4%) did not respond to eltrombopag treatment. For patients above 60 years old (n=68), overall response rate was 89.7% (n=61), and for those above 80 years old (n=12), overall response rate was 83% (n=10). Considering thrombocyte count before treatment, eltrombopag significantly increased platelet count at the 1st, 2nd, 3rd, 4th, and 8th weeks of treatment. As the time required for partial or complete response increased, response to treatment was significantly reduced. The time to reach the maximum platelet levels after treatment was quite variable (1-202 weeks). Notably, the higher the maximum platelet count after eltrombopag treatment, the more likely that side effects would occur. The most common side effects were headache (21.6%), weakness (13.7%), hepatotoxicity (11.8%), and thrombosis (5.9%). Conclusion: Results of the current study imply that eltrombopag is an effective therapeutic option even in elderly patients with chronic ITP. However, patients must be closely monitored for response and side effects during treatment. Since both response and side effects may be variable throughout the follow-up period, patients should be evaluated dynamically, especially in terms of thrombotic risk factors.

Comparison of two minimal invasive techniques of splenectomy: Standard laparoscopy versus transumbilical multiport single-site laparoscopy with conventional instruments.

BACKGROUND: Laparoendoscopic single-site (LESS) splenectomy which is performed on small number of patients, has been introduced with better cosmetic outcome, less postoperative pain, greater patient satisfaction and faster recovery compared to standard laparoscopy. MATERIALS AND METHODS: Thirty six patients were included in the study comparing standard laparoscopic splenectomy (LS, 17 patients) transumbilical multiport splenectomy performed with conventional laparoscopic instruments (TUMP-LS, 19 patients). Two groups of patients were compared retrospectively by means of operation time, intra- and postoperative blood loss, perioperative complications, packed red cell and platelet requirements, lenght of hospitalization, pain scores and patient satisfaction. RESULTS: There was no mortality in any of the groups, and no significant differences determined in operative time (P = 0,069), intraoperative blood loss (P = 0,641), patient satisfaction (P = 0,506), pain scores (P = 0,173) and the average length of hospital stay (P = 0,257). Umbilical incisions healed uneventfully and no hernia formation or wound infection was observed during follow-up period (2-34 months). There were no conversions to open surgery. CONCLUSIONS: Transumbilical multiport splenectomy performed with the conventional laparoscopic instruments is feasible and could be a logical alternative to classical laparoscopic splenectomy by combining the advantages of single access techniques and standard laparoscopy.

Thrombocytosis and small bowel perforation: unusual presentation of abdominopelvic actinomycosis.

Intrauterine devices (IUD) are frequently used as a family planning procedure in developing countries because they are easy to administer and governmental policies support their use in many countries. It is recommended that IUDs be removed or replaced after 10 years, but longer use is common, especially in developing countries. In some cases, rare infections such as pelvic inflammatory diseases, pelvic tuberculosis, or abdominopelvic actinomycosis related to IUD can develop. Pelvic actinomycosis is a rare disease and is often diagnosed incidentally during surgery. In recent years, there has been an increase in actinomycotic infections mostly due to long-term usage of IUD and forgotten intravaginal pessaries. It usually develops as an ascending infection. It is usually associated with non-specific symptoms such as lower abdominal pain, menstrual disturbances, fever, and vaginal discharge. The disease is sometimes asymptomatic. The rate of accurate preoperative diagnosis for pelvic actinomycosis is less than 10%, and symptoms and imaging studies sometimes mimic pelvic malignancy. This report details a case with abdominopelvic actinomycosis associated with an IUD presenting with highly elevated thromboctye count and small bowel perforation with abscess formation.

Incidence of congenital hemolytic anemias in young cholelithiasis patients.

AIM: To clarify the incidence of congenital hemolytic anemias (CHA) in young cholelithiasis patients and to determine a possible screening test based on the results. METHODS: Young cholelithiasis patients (< 35 years) were invited to our outpatient clinic. Participants were asked for comorbidities and family history. The number of gallstones were recorded. Blood samples were obtained to perform a complete blood count, standard Wright-Giemsa staining, reticulocyte count, hemoglobin (Hb) electrophoresis, serum lactate dehydrogenase and bilirubin levels, and lipid profile. RESULTS: Of 3226 cholecystectomy patients, 199 were under 35 years, and 190 with no diagnosis of CHA were invited to take part in the study. Fifty three patients consented to the study. The median age was 29 years (range, 17-35 years), 5 were male and 48 were female. Twelve patients (22.6%) were diagnosed as thalassemia trait and/or iron-deficiency anemia. Hb levels were significantly lower (P = 0.046), and mean corpuscular volume (MCV) and hematocrit levels were slightly lower (P = 0.072 and 0.082, respectively) than normal. There was also a significantly lower number of gallstones with the diagnosis (P = 0.007). CONCLUSION: In endemic regions, for young cholelithiasis patients (age under 35) with 2-5 gallstones, the clinician/surgeon should pay attention to MCV and Hb levels as indicative of CHA.

Cranial involvement in sickle cell disease.

PURPOSE: To evaluate cranial findings in patients with neurologically symptomatic sickle cell disease (SCD). MATERIALS AND METHODS: We studied 50 consecutive patients with SCD and neurologic symptoms. All patients underwent brain MR examinations: all 50 underwent classic MR imaging; 42, diffusion-weighted MR imaging; 10, MR angiography; four, MR venography; and three patients, digital subtraction angiography. RESULTS: Of the 50 SCD patients, 19 (38%) had normal MR findings, and 31 (62%) showed abnormalities on brain MR images. Of the 50 patients, 16 (32%) had ischemic lesions; two (4%), subarachnoid hemorrhage; one (2%), moya-moya pattern; one (2%), posterior reversible encephalopathy; one (2%), dural venous sinus thrombosis; 12 (24%), low marrow signal intensity and thickness of the diploic space; 12 (24%), cerebral atrophy; and two (4%), osteomyelitis. Twenty-seven patients (54%) presented with headache, which was the most common clinical finding. CONCLUSIONS: The cranial involvement is one of the most devastating complications of SCD. Early and accurate diagnosis is important in the management of cranial complications of SCD.

Fluorescent in situ hybridization studies in multiple myeloma.

Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) results of bone marrow samples of 36 multiple myeloma (MM) patients at the time of diagnosis have been evaluated. Three probes for chromosome 13q (RB1, D13S319, D13S25), one for 14q32 (IgH) and one for 17p13 (p53) have been used for hybridization with fixed cells. Twenty patients (55.5%) had normal karyotypes, whereas eight (22.2%) had numerical or structural chromosomal abnormalities. We did not find metaphases for chromosome analysis in eight (22.2%) patients. Fluorescence in situ hybridization analyses revealed at least one or more abnormal results in 25 (69.5%) cases, whereas 11(30.5%) cases had no abnormal findings. 14q32 rearrangement was the most common finding in FISH analyses and has been detected in 21 cases (58.3%). 13q deletion and 17p deletion have been detected in 11 (30.5%) and 5 (13.9%) cases, respectively. Fluorescence in situ hybridization studies including 14q32 and 17p13 chromosome regions may yield quite significant results during clinical follow-up of MM.

A case of disseminated intravascular coagulation caused by Brucella melitensis.

OBJECTIVE: Although the haematological abnormalities were common accompaniments of brucellosis, overt disseminated intravascular coagulopathy was reported only in a case at the literature. We report here a case of Brucella melitensis sepsis which showed an acute onset with clinical and hematological findings disseminated intravascular coagulopathy METHODS: The patient had a physical examination, coagulation screening tests, tests of thrombin generation and fibrinolysis, bone marrow aspirate, serum Brucella agglutination test and blood culture. A case of Brucella infection presenting at the onset as a disseminated intravascular coagulation with gingival bleeding and echimotic lesions on abdomen is reported. A hemogram showed severe thrombocytopenia, anemia, and leukopenia. Anisocytozis, poikilocytozis, shift to the left of the granulocytic series, fragmented red blood cells, toksic granulation, were present in the peripheral smear. Bone marrow aspirate revealed a hypercellular marrow without granulomata or malignant precursors. Prothrombin time: 20 s, activated partial thromboplastin time:53.9 s were found longer than normal. D-dimer: 1056 mug/l (50-228 mug/l) was found. Fibrinogen was too low to detected in serum. Patient had Brucella melitensis isolated from blood cultures. RESULTS: After appropriate antimicrobial therapy, the clinical and hematological status of the patient improved, and 4 days later with disappearance of all hematological abnormalities. CONCLUSISON: Diagnosis of brucellosis may be delayed, particularly if uncommon features such as pancytopenia and disseminated intravascular coagulopathy are present. Hence, brucellosis must be considered in the differential diagnosis of all those conditions leading to pancytopenia and disseminated intravascular coagulopathy in areas endemic for brucellosis.

Platelet P-selectin expression in patients with sickle cell disease who undergo apheresis.

Activated platelets have been identified in patients with sickle cell disease. However, the association of platelet P-selectin expression and automated red cell exchange procedures in these patients is not well known. We hypothesized that altered whole platelet P-selectin expression is associated with automated red cell exchange. Flow cytometric quantification of platelet P-selectin expression was carried out in 23 patients with sickle cell disease before and after automated red cell exchange. P-selectin expression was quantified as a binding index for platelet P-selectin (the percentage of positive platelets multiplied by the mean fluorescence of positive platelets). The patients were divided into two groups: individuals with painful vaso-occlusive crises (four women and five men; group 1) and those in a steady state (six women and eight men; group 2). The 33 exchange procedures were evaluated prospectively and used acid-citrate-dextrose A solution (whole blood to anticoagulant ratio = 14:1). Platelet P-selectin expression did not significantly change after automated red cell exchange. Clinical factors such as the volume of replacement fluid and the citrate infusion rate did not correlate with postapheresis platelet P-selectin expression. In addition, the association of platelet P-selectin expression and automated red cell exchange was independent of other laboratory factors (hematocrit level, hemoglobin S level, platelet count, and nitric oxide level). Finally, the difference between the study groups regarding platelet P-selectin expression before and after apheresis was insignificant. In conclusion, automated red cell exchange procedures do not induce platelet P-selectin expression in patients with sickle cell disease in the steady state or in vaso-occlusive crisis.

Automated red cell exchange procedures in patients with sickle cell disease.

In automated red cell exchange, about 60% of the patient's red blood cells are exchanged via apheresis for those of the donor. We report the outcome of 83 patients with sickle cell anemia (48 women and 35 men; age range, 17-49 years) who underwent a total of 196 apheresis procedures between December 2003 and October 2006 at our institution. We found that automated red cell exchange involving a reduced citrate infusion rate may provide benefit in the prevention or treatment of vaso-occlusive complications in patients with sickle cell disease and may be associated with protean effects on biochemical dynamics.

A dramatic response to rituximab in a patient with resistant thrombotic thrombocytopenic purpura (TTP) who developed acute stroke.

BACKGROUND: Refractory condition can occur in 10-30% of all cases of thrombotic thrombocytopenic purpura despite increased frequency of total plasma exchange. Rituximab can affect the clinical outcome of the refractory cases. However, little is known about usefulness of rituximab on central nervous system involvement mimicking acute ischemic stroke. METHODS: We report the case of a woman with refractory thrombotic thrombocytopenic purpura who developed an acute onset right sided paralysis, dysarthria, and central facial paralysis, suggestive of cerebrovascular accident while under plasma exchange, corticosteroid, and vincristine therapy. RESULTS: After initiation of rituximab (375 mg/m(2) weekly for 4 weeks), a dramatic response occurred and the patient's neurologic function recovered fully within days. Sustained remission was achieved, and the patient was well 1 year after her admission, while she was on azathioprine treatment. CONCLUSION: This report suggests that rituximab can provide a good outcome of the dramatic central nervous system involvement in patients with thrombotic thrombocytopenic purpura.

The apoptosis of blood polymorphonuclear leukocytes in sickle cell disease.

BACKGROUND: The apoptosis of human polymorphonuclear leukocytes (PMNs) in patients with sickle cell disease (SCD) is not well understood. The goal of this study was to examine the apoptosis of PMNs in patients with SCD and in controls. METHODS: Flow cytometric quantitation of PMN apoptosis was performed in 17 patients during and after sickle cell vasoocclusive crisis and in 17 healthy volunteers. Plasma nitric oxide concentrations were also measured in patients with SCD. RESULTS: The mean of annexin-V and annexin-V/PI staining (early and late apoptotic cells) increased to a greater degree in patients with SCD than in healthy controls for patients with SCD during and after vasoocclusive crisis. The mean of PI staining showing dead cells was higher only in patients after SCD crisis than in healthy controls. In the SCD groups during and after vasoocclusive crisis, there was no difference between PMN apoptosis levels. Furthermore, plasma nitric oxide concentrations were not correlated with PMN apoptosis. CONCLUSIONS: There was an evidence that the alteration of blood PMN apoptosis could contribute to the pathogenetic mechanisms of vasoocclusion in patients with SCD. This can be attributed to the effects of numerous inflammatory mediators rather than simply the effects of nitric oxide.

Bone marrow necrosis: a rare complication of herbal treatment with Hypericum perforatum (St. John's wort).

A 22-year-old man presented with fever and ulceration of the oral mucosa. The patient had pharyngeal and esophageal candidiasis. For the 3 weeks prior to presentation, he had been taking approximately 1000 mg/day of flowering herbs (Hypericum perforatum L, known as St. John's wort) for treatment of depression. A complete blood count on the first day of hospitalization showed agranulocytosis and normocytic anemia. His condition worsened, and he developed progressive dysphagia. A bone marrow biopsy on day 3 revealed bone marrow necrosis. After the diagnosis was established (day 3 of hospitalization), treatment with granulocyte colony-stimulating factor 48 U/day, intravenous immunoglobulin 400 mg/kg, and amphotericin B 100 mg/day was initiated. The patient did not respond, and died within one week of the diagnosis. This cases suggests that Hypericum species may lead to severe hematologic toxicity, with conditions involving bone marrow necrosis.

Aplastic anemia in a professional musician exposed to instrument polish.

Chemicals are known to cause toxin-induced aplastic anemia. However, some chemicals documented in only a few cases to possess only a possibility of toxic potential may also be responsible for the development of aplastic anemia. This report presents a case of a string musician with bone marrow failure. The patient used a certain type of polish (komalak) to shine his musical instrument and did this frequently. He presented with nasal bleeding, and a workup on admission revealed pancytopenia. Aplastic anemia was diagnosed on the basis of bone marrow histology results. An analysis for gene polymorphism related to the metabolic detoxification enzymes glutathione S-transferase and N-acetyltransferase 2 indicated that the patient was genetically susceptible to developing toxicity. This case suggests that frequent use of this polish may cause a toxic effect that leads to bone marrow failure. Musicians should be made aware of the risks associated with these types of chemicals.

Partial splenic embolization in myelodysplastic syndrome associated with immune thrombocytopenia.

BACKGROUND: Currently, all treatments for patients with myelodysplastic syndrome (MDS) are still experimental. Supportive care, including treatment of infection and transfusion of blood and blood-related products, remains the most important aspect of therapy in these cases. This article discusses the results of partial splenic embolization in a patient who had MDS with refractory anemia. METHODS: The patient was placed under observation and was given supportive treatment. In addition to MDS with refractory anemia, there were management difficulties related to severe thrombocytopenia after alloimmunization from multiple blood transfusions. Therefore it is thought that partial splenic embolization might be an alternative treatment for the management thrombocytopenia. RESULTS: Although the patient did not live long because of the sepsis and ileus, hematologic response was obtained. CONCLUSION: The observation during the treatment in this case suggests that partial splenic embolization may be a valid new alternative for treating thrombocytopenia due to alloimmunization.