Identification of the molecular etiology in rare congenital hemolytic anemias using next-generation sequencing with exome-based copy number variant analysis.

OBJECTIVES: In congenital hemolytic anemias (CHA), it is not always possible to determine the specific diagnosis by evaluating clinical findings and conventional laboratory tests. The aim of this study is to evaluate the utility of next-generation sequencing (NGS) and clinical-exome-based copy number variant (CNV) analysis in patients with CHA. METHODS: One hundred and forty-three CHA cases from 115 unrelated families referred for molecular analysis were enrolled in the study. Molecular analysis was performed using two different clinical exome panels in 130 patients, and whole-exome sequencing in nine patients. Exome-based CNV calling was incorporated into the traditional single-nucleotide variant and small insertion/deletion analysis pipeline for NGS data in 92 cases. In four patients from the same family, the PK Gypsy variant was investigated using long-range polymerase chain reaction. RESULTS: Molecular diagnosis was established in 86% of the study group. The most frequently mutated genes were SPTB (31.7%) and PKLR (28.5%). CNV analysis of 92 cases revealed that three patients had different sizes of large deletions in the SPTB and six patients had a deletion in the PKLR. CONCLUSIONS: In this study, NGS provided a high molecular diagnostic rate in cases with rare CHA. Analysis of the CNVs contributed to the diagnostic success.

Efficacy and safety of ruxolitinib in patients with myelofibrosis: a retrospective and multicenter experience in Turkey

Background/aim: The aim of this study is to assess the efficacy and safety of ruxolitinib in patients with myelofibrosis. Materials and methods: From 15 centers, 176 patients (53.4% male, 46.6% female) were retrospectively evaluated. Results: The median age at ruxolitinib initiation was 62 (28­87) and 100 (56.8%) of all were diagnosed as PMF. Constitutional symptoms were observed in 84.7%. The median initiation dose of ruxolitinib was 30 mg (10­40). Dose change was made in 69 (39.2%) patients. Forty seven (35.6%) and 20 (15.2%) of 132 patients had hematological and nonhematological adverse events, respectively. The mean spleen sizes before and after ruxolitinib treatment were 219.67 ± 46.79 mm versus 199.49 ± 40.95 mm, respectively (p < 0.001). There was no correlation between baseline features and subsequent spleen response. Overall survival at 1-year was 89.5% and the median follow up was 10 (1­55) months. We could not show any relationship between survival and reduction in spleen size (p = 0.73). Conclusion: We found ruxolitinib to be safe, well tolerated, and effective in real-life clinical practice in Turkey. Ruxolitinib dose titration can provide better responses in terms of not only clinical benefit but also for long term of ruxolitinib treatment.

The Turkish experience with therapeutic plasma exchange: A national survey.

Therapeutic plasma exchange (TPE) is used to treat more than 60 diseases worldwide and has drawn growing interest. Little is known about the current situation of TPE activity in Turkey, so we developed a survey to obtain information about this timely topic. We collected data on TPE from 28 apheresis units throughout Turkey. We performed a total of 24,912 TPE procedures with 3203 patients over the past decade. Twenty years ago, the majority of procedures were performed for neurological and hematological disorders, and today, most TPE procedures are done for the same reasons. The only historical change has been an increase in TPE procedures in renal conditions. Currently, renal conditions were more frequently an indication for TPE than rheumatic conditions. Fresh frozen plasma was the most frequently used replacement fluid, followed by 5% albumin, used in 57.9% and 34.6% of procedures, respectively. The most frequently used anticoagulants in TPE were ACD-A and heparin/ACD-A, used with 1671 (52.2%) and 1164 (36.4%) patients, respectively. The frequency of adverse events (AEs) was 12.6%. The most common AEs were hypocalcemia-related symptoms, hypotension, and urticaria. We encountered no severe AEs that led to severe morbidity and mortality. Overall, more than two thirds of the patients showed improvement in the underlying disease. Here, we report on a nationwide survey on TPE activity in Turkey. We conclude that there has been a great increase in apheresis science, and the number of TPE procedures conducted in Turkey has increased steadily over time. Finally, we would like to point out that our past experiences and published international guidelines were the most important tools in gaining expertise regarding TPE.

Efficacy and safety of lenalidomide and dexamethasone in patients with relapsed/ refractory multiple myeloma: a real-life experience

Background/aim: In Turkey, lenalidomide plus dexamethasone (RD) has been used to treat relapsed/refractory multiple myeloma (RRMM) since 2010. This retrospective, single-center study evaluated the efficacy and tolerability of RD in patients with RRMM between October 2010 and June 2016. Materials and methods: Patients' records were reviewed, and overall (OS) and progression-free survival (PFS) were assessed. Results: One hundred and twenty patients (71 males; 59.2%) were included in the study. The median number of prior lines of treatment was one (1­4); 72 patients (60.0%) received RD as second-line therapy and 51 patients (42.5%) had previously undergone autologous stem cell transplantation (ASCT). The overall response rate was 72.5%, with 19% of these patients achieving a complete response. The median length of follow-up and duration of response to RD was 14 months and 19 months, respectively. Median OS and PFS were 32 and 21 months, respectively. Prior ASCT, an overall response, and treatment with RD for >12 cycles were identified as independent prognostic factors for OS and PFS. Adverse events (AEs) occurred in 69 (57.5%) and 14 patients (11.7%) discontinued treatment due to AEs. Conclusion: We found RD to be safe, well tolerated, and effective in RRMM in everyday clinical practice in Turkey.

Risk factors for poor mobilization in solid tumors: How effectively can we mobilize patients with solid tumors?

BACKGROUND: In the literature, risk factors for poor mobilization were tried to identify. However, most of the studies consisted heterogeneous group of patients including both hematologic and oncologic malignancies. In this study, we aimed to identify the risk factors for poor mobilization in adults with solid tumors. METHODS: We enrolled 49(47 men, 2 women) adult patients with solid tumor who were mobilized between September 2007 and February 2017. All the mobilization procedures were performed with G-CSF(10µg/kg/day) with chemotherapy. Mobilization insufficiency was defined as peripheral blood CD34+stem cell number less than 10/µl and/or total collected CD34+cells less than 2.5×10 6/kg. RESULTS: The patients were divided into two groups, patients with successful mobilization at the first attempt(group 1, 36 patients,73.5%) and poor mobilizers (group 2, 13 patients 26.5%). Second and third mobilization attempt was needed in 11 and 2 patients, respectively. The median number of CD34+cells collected was 7,08×106/kg(0,6-19) with a median 4(1-6) apheresis. There was no statistical difference between two groups in terms of patient's and mobilization characteristics. Only number of CD 34+stem cells collected was statistically different (median 9,07×106/kg CD34+cells in group 1 versus 2,14×106/kg in group 2, p<0.05). The only possible risk factor that we could define was presence of organ metastasis. CONCLUSIONS: Since several methods and new drugs are available for peripheral stem cell collecting, risk factors should be identified clearly in adult population with solid tumors. So multicenter studies should be constructed for resolving this problem.

Epidemiology and analysis of invasive fungal infections in patients withhematological malignancies: a single-center real-life experience

Background/aim: Invasive fungal infection (IFI) causes morbidity and mortality among patients with hematological malignancies. We evaluated the incidence and treatment characteristics of IFIs between October 2012 and December 2013. Materials and methods: Patients who received chemotherapy or stem cell transplantation were retrospectively evaluated. Fungal infections were classified according to EORTC criteria.Results: Prophylaxis and antifungal therapy were given in 30.5% and 23.6% of 522 chemotherapy courses, respectively. The incidence of proven/probable IFI was 6.7%. The incidence of IFI among patients who received prophylaxis was significantly higher than among those who did not receive it (11.3% vs. 4.6%, P = 0.005). There was no significant difference between patients who received mold-active and no mold-active prophylaxis (P = 0.098). The most common single agent therapy and causative pathogen was liposomal amphotericin B (57.1%) and Aspergillus (n = 5), respectively. IFI-attributable mortality rate was 14.2% in 6 weeks.Conclusion: The IFI incidence and mortality rate were similar to that reported in the literature. The IFI rate was higher in the group using prophylaxis, as this is a high-risk group. Although the IFI rate was not significantly different between groups using prophylaxis, patients should be followed closely for the effective use of posaconazole prophylaxis.

Adult Onset Langerhans Cell Histiocytosis: Clinical Characteristics and Treatment Outcomes.

PURPOSE: Langerhans cell histiocytosis (LCH) is a rare disease that can affect all tissues and organs. Our study evaluated the clinical characteristics and treatment outcomes of adult-onset LCH patients in a tertiary center. MATERIALS AND METHODS: Adult patients diagnosed with LCH were retrospectively evaluated. Their initial symptoms, stratification according to disease involvement, treatment details, treatment responses, and overall and progression-free survival (PFS) were analyzed. RESULTS: Thirty-three patients were included. There were 21 single system LCH, 10 multisystem LCH, and 2 pulmonary LCH patients. Patients with single system unifocal involvement were successfully treated with local therapies such as surgery and radiotherapy. Most of the multisystem LCH patients and patients with single system multifocal involvement were treated with systemic chemotherapy. Cladribine was the first choice in 10 out of 11 patients who received chemotherapy. Among all patients, the overall response rate (ORR) was 97%. Among those who had cladribine in the first-line the ORR was 81%. All these patients achieved a complete remission and were alive at the last visit. The median follow-up was 38 (range, 2-183) months. The median PFS has not yet been reached. Ten-year PFS was 90.9%. CONCLUSION: Besides successful local treatments with surgery and radiotherapy, our study provides information for front-line cladribine treatment.

Factor V G1691A (Leiden) and prothrombiG20210A gene mutation status, and thrombosis in patients with chronic myeloproliferative disorders.

OBJECTIVE: The aim of this study was to examine Factor V G1691A (Leiden) (FVL) and prothrombin G20210A (PT) gene mutation status, and their relationship with thrombosis in patients with chronic myeloproliferative disorders (CMPDs). METHODS: The study included 160 patients with a CMPD that were regularly followed-up between 1993 and 2009. FVL and PT mutation status was established based on blood samples analyzed via PCR using specific primers. RESULTS: The frequency of FVL and PT mutation was 12.5% and 4.4%, respectively. In total, 27 episodes of thrombosis occurred in 24 (15%) of the patients, and there wasn't an association between the observed thrombotic events, and FVL or PT mutations. Hepatic vein thrombosis was noted in 3 patients that had FVL mutation, of which 1 also had PT mutation. CONCLUSION: We did not observe a relationship between thrombosis, and FVL or PT mutations in CMPD patients; however, 3 of the patients that had hepatic vein thrombosis also had FVL mutation. Larger studies are needed to more clearly determine if all CMPD patients with hepatic vein thrombosis need be investigated for FVL and PT mutation.

The Effect of Cryotherapy on the Prevention of Oral Mucositis and on the Oral pH Value in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of cryotherapy on the prevention of oral mucositis (OM) and on the oral pH value in patients with multiple myeloma undergoing autologous stem cell transplantation. DATA SOURCES: This nonrandomized controlled clinical trial was carried out in Bone Marrow Transplant Centers of three hospitals with total 32 patients. In addition to standard oral care, a total of 80 minutes of cryotherapy was applied to the experimental group. OM was assessed according to the World Health Organization's Oral Toxicity Scale before chemotherapy and for 21 days after chemotherapy (every day in the first 14 days, then every other day until the 21st day [if not discharged]). CONCLUSION: According to the findings, cryotherapy did not change the incidence of oral OM, and neither affected the severity of nor decreased the duration of it. Oral pH value was found to be significantly different between the patient groups only before and 1 day after chemotherapy. IMPLICATIONS FOR NURSING PRACTICE: Cryotherapy is an inexpensive, easy-to-use method with no side effects; it would be beneficial to continue cryotherapy to prevent the development of OM in patients with cancer receiving drugs with a short half-life such as melphalan. It is also recommended to conduct further studies with different chemotherapy drugs with short half-lives to determine its effect on the prevention of OM development.

In Vitro Closure Times (PFA-100) Are Different Between Peritoneal Dialysis and Hemodialysis.

INTRODUCTION: Platelet dysfunction is not uncommon in patients with end-stage renal disease (ESRD). Type of renal replacement therapy may have an effect on platelet functions, which has not been well investigated. We evaluated in vitro closure time (CT) differences between peritoneal dialysis (PD) and hemodialysis (HD) patients using platelet function analyzer (PFA-100)and observed a significant difference between these renal replacement therapies. METHODS: Patients with ESRD undergoing PD (n = 24) or HD (n = 23) for more than 6 months were included. Blood samples for collagen/epinephrine (Col/EPI) and collagen/adenosine diphosphate (Col/ADP) measurements were obtained before HD at a mid-week session for HD patients and at an outpatient control time for PD patients. RESULTS: Three of 24 (12.5%) PD patients and 16 of 23 (69.5%) HD patients had prolonged PFA-100 Col/EPI, p< 0.001. Likewise, 4.2% of PD patients and 87.0% of HD patients had prolonged PFA-100 Col/ADP, p< 0.001. Moreover, the median times of PFA-Col/EPI and PFA-100 Col/ADP were significantly lower in PD patients compared with those of HD patients (p< 0.001). Multivariate analysis showed that the type of renal replacement was a risk factor for both elevated PFA-100 Col/ADP and PFA-100 Col/EPI after adjusted for platelets, hematocrit, and urea (p< 0.001). CONCLUSIONS: The type of renal replacement therapy may have an effect on in vitro CTs; therefore, studies including more patients with long-term follow-up are needed to investigate if the difference has any impact on clinical outcomes.

Hemophagocytic syndrome with erythrocyte phagocytosis by the myeloid precursors in a patient with AML -M2.

Hemophagocytic syndrome is characterized by fever, fatigue, weight loss, lymphadenopathy, and laboratory abnormalities including pancytopenia, liver dysfunction, hypertriglyceridemia and hyperfibrinemia. Histopathologically, lesions are characterized by mononuclear cell infiltration with marked histiocyte proliferation and phagocytosis of erythrocytes, leukocytes, platelets and their precursors by activated macrophages in the reticuloendothelial tissues. Hemophagocytic syndrome may develop from strong immunological stimuli such as severe infection, malignancy and autoimmune diseases. We present a 73-year-old man with acute myeloblastic leukemia FAB M2 type (AML M2) whose bone marrow histology showed unusual hemophagocytosis by myeloid cells and myeloblasts.

A Retrospective Comparison of TECAM and BEAM Conditioning Regimens Before Autologous Hematopoietic Stem Cell Transplant in Lymphoma Patients: Efficacy and Toxicity.

OBJECTIVES: The aim of our study was to evaluate the efficacy and toxicity of TECAM (thiotepa, etoposide, cyclophosphamide, cytarabine, and melphalan) and BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning regimens before autologous hematopoietic stem cell transplant in patients with lymphoma. MATERIALS AND METHODS: We retrospectively analyzed 108 relapsed/refractory lymphoma patients who had high-dose treatments followed by autologous hematopoietic stem cell transplant between October 2012 and February 2017. RESULTS: At a median follow-up period of 16 months, the estimated 2-year progression-free survival rates for the TECAM and BEAM groups were 55.7% and 52.9%, respectively (P = .811). The estimated 2-year overall survival rate in the TECAM group (55.9%) was relatively inferior to that shown in the BEAM group (67%), but the differences were not significant (P = .238). No differences were observed for time to hematopoietic recovery and duration of hospitalization. Incidences of transplant-related infectious and noninfectious complications were similar for each conditioning regimen. CONCLUSIONS: Our experience shows that the TECAM regimen is an effective high-dose chemotherapy for lymphoma patients before autologous hematopoietic stem cell transplant.

Carfilzomib experience in relapsed/refractory multiple myeloma: a single-center experience

Background/aim: Carfilzomib (CFZ) is a new-generation proteasome inhibitor with significant activity in relapsed or refractory multiple myeloma (R/R-MM). We have retrospectively evaluated R/R-MM patients who were treated with CFZ plus dexamethasone. Materials and methods: Twenty-one R/R-MM patients who were treated with CFZ plus dexamethasone between October 2013 and January 2016 were screened. The patients were followed until March 2016 after CFZ treatment. Results: Ten (47.6%) of the patients were female and 11 (52.4%) of them were male. The median age was 62 (47-76) years. The median number of prior treatment lines was 3 (2-7). The median number of administered cycles of treatment for CFZ was 4 (1-10). The median overall response rate was 26.3%. The most common hematological adverse events were anemia and thrombocytopenia (38%). The most common nonhematological adverse event was fatigue (71.4%). One patient died because of a cerebrovascular event and 1 patient died because of pneumonia during the treatment period. The median duration of response rate and time to next therapy were 8 (7-9) and 3 (2-16) months, respectively. The median overall survival was 8 (0.5-33) months. Conclusion: Despite the small number of patients, our results suggest that CFZ provides acceptable responses in heavily pretreated R/R-MM patients.

Molecular weight dependent tissue factor pathway inhibitor release by heparin and heparin oligosaccharides.

Heparin and low molecular weight heparins exert their vascular effects by mobilizing tissue factor pathway inhibitor (TFPI) from the vascular endothelium into the blood circulation. We compared the influence of molecular weight on the TFPI release by heparin and its fractions in a non-human primate model. Primates were treated with unfractionated heparin, a low molecular weight heparin (gammaparin), or a heparin-derived oligosaccharide mixture (C3). Endothelial TFPI release was determined using both immunologic and functional assays. After intravenous administration, all agents significantly increased TFPI levels (p<0.05) in a dose dependent manner. The increase produced by unfractionated heparin and gammaparin was greater than that by C3 at an equal dosage (p<0.05). With subcutaneous injection, all agents produced less TFPI release. Repeated administration of heparin-derived oligosaccharides gradually increased TFPI release. A 1.89 fold increase in TFPI levels was observed 4 days after C3 treatment (2.5 mg/kg). Our findings indicated that TFPI release is dependent on the molecular weight of heparin and its derivatives. Heparin oligosaccharides exert their vascular effects through increased TFPI release after long-term repeated administration.

Progressive Transformation of Germinal Centers: Single-Center Experience of 33 Turkish Patients.

Progressive transformation of germinal centers (PTGCs) is a benign disease of the lymph nodes that is rarely associated with Hodgkin disease. We reviewed the clinical and pathologic features of PTGCs and the relationship of PTGCs with lymphoid neoplasia in an adult population. The data from 33 patients who were diagnosed with PTCGs were retrospectively analyzed. Of the 33 PTGC patients, 48.5% were men and 51.5% were women, with a mean age of 43.8 years at diagnosis. Most of the enlarged and excised lymph nodes were cervical and axillary. Diffuse large B-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma was detected concurrent with PTGC in 2 patients. Also, PTGCs was detected 3 years after the diagnosis of diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and T-cell-rich B-cell lymphoma in 3 patients. No relapse was found in the patients with lymphoma, and no progression to lymphoma was detected during the follow-up of the other patients. PTGCs is not considered a premalignant entity; however, the development of lymphoma has been reported rarely. If PTGCs occurs in the follow-up process of patients with lymphoma, the follow-up intervals should be shortened.

Pesg PNH diagnosis, follow-up and treatment guidelines.

PNH Education and Study Group (PESG) have been established in December 2013 as a non-profit, independent, medical organization www.pesg.org. Paroxysmal Nocturnal Hemoglobinuria (PNH) is a multi-systemic disease that should be treated with a multidisciplinary approach. Patients may apply to the clinics other than the hematology due to variability and diversity of clinical findings which lower the rate of diagnosis due to low awareness about PNH. PNH might be overlooked and diagnosis might be delayed. Regarding these, PESG was established with the collaboration of Immunology, Cardiology, Thorax Diseases (Pulmonology), Neurology, Gastroenterology, General Surgery and Urology specialists in addition to hematologists dealing with PNH. The PESG study group aims to increase the awareness about PNH, including training activities about PNH, strengthening the relations between clinics and planning of clinical studies as a goal. It is the first professional organization focusing on PNH, in Turkey.In this guideline, we want to facilitate the diagnosis attributes of physicians from all specializations that deal with PNH and its systemic complications. One can perceive this as a tailor made guideline of international guidelines but not a compilation.

Antithrombotic agents in the treatment of severe sepsis.

Sepsis, a systemic inflammatory syndrome, is a response to infection and when associated with multiple organ dysfunction is termed severe sepsis. It remains a leading cause of mortality in the critically ill. The response to the invading microorganisms may be considered as a balance between a pro-inflammatory and an anti-inflammatory reaction. While an inadequate pro-inflammatory reaction and a strong anti-inflammatory response could lead to overwhelming infection and the death of the patient, a strong and uncontrolled pro-inflammatory response, manifested by the release of pro-inflammatory mediators may lead to microvascular thrombosis and multiple organ failure. Endotoxin triggers sepsis via the release of various mediators such as tumour necrosis factor-alpha and interleukin-1 (IL-1). These cytokines activate the complement and coagulation systems, release adhesion molecules, prostaglandins, leukotrienes, reactive oxygen species and nitric oxide. Other mediators involved in the sepsis syndrome include IL-1, -6 and -8; arachidonic acid metabolites; platelet activating factor; histamine; bradykinin; angiotensin; complement components and vasoactive intestinal peptide. These pro-inflammatory responses are counteracted by IL-10. Most of the trials targeting the different mediators of the pro-inflammatory response have failed due to a lack of correct definition of sepsis. Understanding the exact pathophysiology of the disease will enable more advanced treatment options. Targeting the coagulation system with various anticoagulant agents including, activated protein C, and tissue factor pathway inhibitor (TFPI) is a rational approach. Many clinical trials have been conducted to evaluate these agents in severe sepsis. While trials on antithrombin and TFPI were not so successful, the double-blind, placebo-controlled, Phase III trial of recombinant human activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) was successful, creating a significant decrease in mortality when compared to the placebo group. A better understanding of the pathophysiologic mechanism of severe sepsis will provide better treatment options, and combination antithrombotic treatment may provide a multipronged approach for the treatment of severe sepsis.

Anti-Xa and anti-IIa drugs alter international normalized ratio measurements: potential problems in the monitoring of oral anticoagulants.

Several of the newly developed anti-Xa and anti-IIa agents have been shown to influence the International Normalized Ratio (INR) values. During phase I trials with normal healthy volunteers and phase II study patients who were given warfarin and concomitant anti-IIa or anti-Xa agents, it has been reported that INR values were falsely elevated. It is of critical importance to know of the effects of these agents on INR to avoid dosage errors. To study the influence of these agents on INR, we used several anti-IIa agents (argatroban, recombinant hirudin, efegatran, and PEG-hirudin) and anti-Xa drugs (pentasaccharides such as fondaparinux and idraparinux, DX-9065a and JTV-803). The anti-IIa drugs were supplemented in citrated plasma at a concentration of 0 to 1 microg/mL level and anti-Xa drugs in the range of 0 to 25 microg/mL. The IC(50) values for each of these agents were calculated. Four different commercially available prothrombin time (PT) reagents were used to perform the PT assays and to calculate the relative INR values. Direct synthetic factor IIa and Xa inhibitors exhibited a concentration-dependent increase in the INR values. Hirudin, efegatran, and PEG-hirudin showed a weaker effect, whereas argatroban showed a much higher elevation of the INR values. Synthetic indirect anti-Xa agents such as the pentasaccharide did not show any effect on the INR values. Furthermore, prothrombin time reagents with high ISI values exhibited disproportionally higher INR values for both the direct anti-Xa and anti-IIa agents. Elevation of INR values has therapeutic implications when non-oral anticoagulant drugs are used in combination with drugs such as warfarin. Because of the false elevation of INR values with some of the non-oral anticoagulant drugs, patients who are on concomitant warfarin therapy should be carefully evaluated for their corresponding INR values for proper dosing. To avoid dosing errors it is best not to use the INR values in the therapeutic monitoring of anti-Xa and anti-IIa agents either in the monotherapeutic or polytherapeutic modalities. These data also warrant the development clinically relevant methods for the monitoring of the concomitant use of newly developed anti-Xa and anti-IIa drugs with oral anticoagulants.