Optical Genome Mapping as an Alternative to FISH-Based Cytogenetic Assessment in Chronic Lymphocytic Leukemia.

The fluorescence in situ hybridization (FISH) technique plays an important role in the risk stratification and clinical management of patients with chronic lymphocytic leukemia (CLL). For genome-wide analysis, FISH needs to be complemented with other cytogenetic methods, including karyotyping and/or chromosomal microarrays. However, this is often not feasible in a diagnostic setup. Optical genome mapping (OGM) is a novel technique for high-resolution genome-wide detection of structural variants (SVs), and previous studies have indicated that OGM could serve as a generic cytogenetic tool for hematological malignancies. Herein, we report the results from our study evaluating the concordance of OGM and standard-of-care FISH in 18 CLL samples. The results were fully concordant between these two techniques in the blinded comparison. Using in silico dilution series, the lowest limit of detection with OGM was determined to range between 3 and 9% variant allele fractions. Genome-wide analysis by OGM revealed additional (>1 Mb) aberrations in 78% of the samples, including both unbalanced and balanced SVs. Importantly, OGM also enabled the detection of clinically relevant complex karyotypes, undetectable by FISH, in three samples. Overall, this study demonstrates the potential of OGM as a first-tier cytogenetic test for CLL and as a powerful tool for genome-wide SV analysis.

The Standard Point-of-Care Hemochron Jr. ACT+ Test in Monitoring Heparin Administration for Cardiopulmonary Bypass in Severe Factor XII Deficiency.

Coagulation factor XII (FXII) is a plasma serine protease that belongs to the contact activation complex responsible for initiating the intrinsic coagulation pathway. FXII deficiency is a rare congenital disorder that is not associated with an increased tendency for bleeding. However, as contact activation is impaired in FXII deficiency, both the celite- and kaolin-initiated activated clotting time (ACT) measurements are prolonged markedly, which poses a challenge for anticoagulation monitoring in patients undergoing cardiac surgery. The authors successfully have used the standard Hemochron Jr. ACT+ test, which is activated by silica and phospholipid in addition to kaolin, to monitor anticoagulation for cardiopulmonary bypass in two patients with severe FXII deficiency. The ACT+ test showed low baseline values, increased adequately in response to heparin, and decreased to baseline after protamine. Importantly, there was no abnormal intra- or postoperative bleeding nor any thrombotic complications. Furthermore, in vitro dose-response ACT+ testing of FXII-deficient blood with increasing heparin concentrations supports the use of ACT+ in FXII deficiency.

Autograft cellular composition and outcome in myeloma patients: Results of the prospective multicenter GOA study.

BACKGROUND: Autologous stem cell transplantation (auto-SCT) is a widely used treatment option in multiple myeloma (MM) patients. The optimal graft cellular composition is not known. STUDY DESIGN AND METHODS: Autograft cellular composition was analyzed after freezing by flow cytometry in 127 MM patients participating in a prospective multicenter study. The impact of graft cellular composition on hematologic recovery and outcome after auto-SCT was evaluated. RESULTS: A higher graft CD34+ cell content predicted faster platelet recovery after auto-SCT in both the short and long term. In patients with standard-risk cytogenetics, a higher graft CD34+ count (>2.5 × 106 /kg) was linked with shorter progression-free survival (PFS; 28 vs. 46 months, p = 0.04), but there was no difference in overall survival (OS) (p = 0.53). In a multivariate model, a higher graft CD34+ CD133+ CD38- (>0.065 × 106 /kg, p = 0.009) and NK cell count (>2.5 × 106 /kg, p = 0.026), lenalidomide maintenance and standard-risk cytogenetics predicted better PFS. In contrast, a higher CD34+ count (>2.5 × 106 /kg, p = 0.015) predicted worse PFS. A very low CD3+ cell count (≤20 × 106 /kg, p = 0.001) in the infused graft and high-risk cytogenetics remained predictive of worse OS. CONCLUSIONS: Autograft cellular composition may impact outcome in MM patients after auto-SCT. More studies are needed to define optimal graft composition.

Autograft cellular composition and outcome in NHL patients: results of the prospective multicenter GOA study.

Autologous stem cell transplantation (auto-SCT) is an established treatment option in patients with non-Hodgkin lymphoma (NHL). In this prospective multicenter study, the effect of infused blood graft cellular composition on post-transplant outcome was analyzed in 129 NHL patients. Higher graft CD34+ cell content (>2.5 × 106/kg) correlated with better progression-free survival (PFS) (p=.009) and overall survival (OS) (p=.004). Higher graft CD34+CD133+CD38- counts (>0.08 × 106/kg) were also linked with better PFS (p=.03) and OS (p=.004), and these survival benefits retained in multivariate analyses. Higher infused CD3+CD4+ cell count (>37 × 106/kg) predicted better PFS (p=.013) and OS (p=.007) in multivariate analysis. Autograft cellular composition seems to impact outcome in NHL patients. These observations regarding composition of optimal graft in autologous setting should be validated in an independent patient series or in a randomized study.

CD34+ cell mobilization, blood graft composition, and posttransplant recovery in myeloma patients compared to non-Hodgkin's lymphoma patients: results of the prospective multicenter GOA study.

BACKGROUND: Autologous stem cell transplantation is an established treatment option for patients with multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL). STUDY DESIGN AND METHODS: In this prospective multicenter study, 147 patients with MM were compared with 136 patients with NHL regarding the mobilization and apheresis of blood CD34+ cells, cellular composition of infused blood grafts, posttransplant recovery, and outcome. RESULTS: Multiple myeloma patients mobilized CD34+ cells more effectively (6.3 × 106 /kg vs. 3.9 × 106 /kg, p = 0.001). The proportion of poor mobilizers (peak blood CD34+ cell count <20 × 106 /L) was higher in NHL patients (15% vs. 3%, p < 0.001). Plerixafor was added to rescue the mobilization failure in 17 MM patients (12%) and in 35 NHL patients (26%; p = 0.002). The infused grafts contained more natural killer (NK) and CD19+ cells in MM patients. Blood platelet and NK-cell counts were higher in MM patients posttransplant. Early treatment-related mortality was low in both groups, but NHL patients had a higher late (>100 days) nonrelapse mortality (NRM; 6% vs. 0%, p = 0.003). CONCLUSIONS: Non-Hodgkin's lymphoma and MM patients differ in terms of mobilization of CD34+ cells, graft cellular composition, and posttransplant recovery. Thus, the optimal graft characteristics may also be different.

Hyperthermic intraperitoneal chemotherapy enhances blood coagulation perioperatively evaluated by thromboelastography: a pilot study.

Background and Objectives: Postoperative thromboembolism is a significant cause of prolonged recovery in patients undergoing cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Thromboelastography (TEG) can detect hypercoagulable states and predict thromboembolic complications after surgery. This study assessed the impact of CRS and HIPEC on TEG values.Methods: TEG parameters reaction time (R), kinetics time (K), angle (α), maximum amplitude (MA), and lysis percent at 60 min (LY60) were determined preoperatively, and at the end of CRS, during HIPEC, and at the end of the operation using blood samples from 15 HIPEC patients. Platelets, P-TT, and aPTT were also determined before and after CRS.Results: A total of 75 samples were analyzed. During CRS, there was a significant reduction in the mean MA (3.06 mm, p = 0.001). The mean P-TT declined by 32% (p < 0.001) and mean platelets by 55 × 109/L (p < 0.001). During HIPEC, the mean R and K shortened by 1.04 min (p = 0.015) and 0.18 min (p = 0.018), respectively, whereas α increased by 2.48° (p = 0.005).Conclusions: During CRS, both TEG and conventional laboratory tests indicated hypocoagulation. During HIPEC, however, the initiation of coagulation and the kinetics of thrombin formation were accelerated.

Blood graft composition and post-transplant recovery in myeloma patients mobilized with plerixafor: a prospective multicenter study.

The composition of autologous blood grafts after cryopreservation, post-transplant hematological recovery up to 1 year and immune recovery up to 6 months as well as outcome was analyzed in 87 patients with multiple myeloma (MM). The patients receiving added plerixafor due to poor mobilization (11%) were compared to those mobilized with G-CSF or cyclophosphamide (CY) plus G-CSF. The use of plerixafor was found to significantly affect the graft composition as there was a significantly higher proportion of the more primitive CD34+ cells, higher number of T and B lymphocytes as well as NK cells in the grafts of patients who received also plerixafor. The hematological recovery after auto-SCT was comparable between the groups. The recovery of CD3+CD4+ T cells was faster in plerixafor mobilized patients at 1 and 3 months post-transplant. There were no significant differences in progression-free (PFS) or overall survival (OS) according to the plerixafor use.

Thromboelastography Values Remain Hypercoagulative 6 Months After Obesity Surgery: a Pilot Study.

PURPOSE: Obesity causes a prothrombotic state and is known as a predisposing factor for thromboembolic events. In this pilot study, we assessed the impact of surgery for obesity and the subsequent weight loss on blood coagulation using traditional coagulation tests and thromboelastography (TEG). MATERIAL AND METHODS: We studied blood samples from 18 patients receiving bariatric surgery. Besides traditional blood coagulation tests and high-sensitivity C-reactive protein (hsCRP) as a marker of inflammation, the TEG parameters reaction time (R), kinetics time (K), angle (α), maximum amplitude (MA), clot strength (G), and lysis percent at 60 min (LY60) were determined preoperatively and on the first postoperative day and 6 months after surgery. RESULTS: Altogether, 54 samples were analyzed. The median MA (71.3 mm), G (12,403.3 d/sc), and hsCRP (3.5 mg/l) were elevated preoperatively. The median hsCRP further increased on the first day postoperatively, but declined to the normal range 6 months after surgery, while MA and G remained elevated. In traditional coagulation tests, there was an increase in median fibrinogen and D-dimer postoperatively. D-dimer normalized (0.4 mg/l) during the study period, while the fibrinogen level (4.1 g/l) remained above the upper limit of normal. CONCLUSIONS: Measured by TEG, patients receiving bariatric surgery have hemostatic abnormalities indicating hypercoagulation at the 6-month follow-up visit, suggesting an elevated risk for thromboembolic events for at least 6 months after surgery.

Neutrophil CD64, C-reactive protein, and procalcitonin in the identification of sepsis in the ICU - Post-test probabilities.

PURPOSE: We were interested in whether C-reactive protein (CRP) and procalcitonin (PCT) distinguish sepsis from non-septic controls and whether a combination of CRP, PCT, and neutrophil CD64 improves identification of sepsis in the intensive care unit (ICU). MATERIALS AND METHODS: We analyzed the CRP and PCT concentrations from 27 patients with sepsis and 15 ICU controls. In addition, CD64 on neutrophils was measured using quantitative flow cytometry. We present a multiple marker analysis for sepsis diagnostics combining neutrophil CD64, CRP, and PCT using post-test analysis. RESULTS: The CRP and PCT values separated sepsis and non-septic ICU patients. In post-test analysis, CRP provided a positive probability of 0.48 and a negative probability of 0.053 for sepsis in the ICU; while, the corresponding values were 0.35 and 0.0059, respectively, for PCT and 0.62 and 0.0013, respectively, for neutrophil CD64. When neutrophil CD64 was analyzed with PCT and CRP, the probabilities were 0.98 and <0.001, respectively. CONCLUSIONS: Neutrophil CD64 expression was superior to PCT and CRP for the identification of sepsis in ICU. Positive post-test probability for any combinations of simultaneously analyzed CRP, PCT and CD64 showed improved diagnostic accuracy for sepsis. This approach may be useful for guiding antibiotic treatment in ICU.

Expression of a specific extracellular matrix signature is a favorable prognostic factor in acute myeloid leukemia.

Relapse of acute myeloid leukemia (AML) is still dramatically frequent, imposing the need for early markers to quantify such risk. Recent evidence point to a prominent role for extracellular matrix (ECM) in AML, but its prognostic value has not yet been investigated. Here we have investigated whether the expression of a 15-ECM gene signature could be applied to clinical AML research evaluating a retrospective cohort of 61 AML patients and 12 healthy donors. Results show that patients whose ECM signature expression is at least twice as that of healthy donors have considerably longer relapse-free survival, with further stage-specific therapy outcomes.

Intrauterine growth restriction and placental gene expression in severe preeclampsia, comparing early-onset and late-onset forms.

OBJECTIVE: To evaluate placental gene expression in severe early- or late-onset preeclampsia with intrauterine growth restriction compared to controls. STUDY DESIGN: Chorionic villus sampling was conducted after cesarean section from the placentas of five women with early- or late-onset severe preeclampsia and five controls for each preeclampsia group. Microarray analysis was performed to identify gene expression differences between the groups. RESULTS: Pathway analysis showed over-representation of gene ontology (GO) biological process terms related to inflammatory and immune response pathways, platelet development, vascular development, female pregnancy and reproduction in early-onset preeclampsia. Pathways related to immunity, complement and coagulation cascade were overrepresented in the hypergeometric test for the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Ten genes (ABI3BP, C7, HLA-G, IL2RB, KRBOX1, LRRC15, METTL7B, MPP5, RFLNB and SLC20A) had a ≥±1 fold expression difference in severe early-onset preeclampsia group compared to early controls. There were 362 genes that had a ≥±1 fold expression difference in severe early-onset preeclampsia group compared to late-onset preeclampsia group including ABI3BP, C7, HLA-G and IL2RB. CONCLUSION: There are significant differences in placental gene expression between severe early- and late-onset preeclampsia when both are associated with intrauterine growth restriction. ABI3BP, C7, HLA-G and IL2RB might contribute to the development of early form of severe preeclampsia.

Monocyte and lymphocyte surface molecules in severe sepsis and non-septic critically ill Patients.

The aim of the present study was to investigate whether expression of monocyte and lymphocyte surface molecules differs between patients with severe sepsis and non-septic patients treated in the intensive care unit (ICU). The expression of monocyte CD14, CD40, CD80 and HLA-DR, and lymphocyte CD69 were analyzed using quantitative flow cytometry on three consecutive days in 27 patients with severe sepsis and in 15 non-septic patients. Receiver operating characteristic analyses were performed and each corresponding area under the curve (AUC) was determined. The results showed that the expression levels of CD40 on monocytes and CD69 on CD4+ T cells and on natural killer (NK) cells were highest in patients with severe sepsis (p < 0.05). Monocyte CD40 and NK cell CD69 expression levels were higher in patients with severe sepsis and positive blood culture compared with those with negative blood culture (p < 0.05). The highest values of AUC for severe sepsis detection were 0.836 for CD40, 0.872 for CD69 on NK cells, and 0.795 for CD69 on CD4+ T cells. These findings suggest that monocyte CD40 and CD69 on NK cells and CD4+ T cells could prove useful for new approaches in the identification of severe sepsis in the ICU.

Cost analysis of a randomized stem cell mobilization study in multiple myeloma.

Upfront autologous stem cell transplantation (ASCT) is the standard therapy for younger multiple myeloma (MM) patients. MM patients usually undergo stem cell mobilization with cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF), or with G-CSF alone. A limited number of randomized studies are available comparing costs of different mobilization strategies. Eighty transplant-eligible patients aged up to 70 years with untreated MM were included in this prospective study. The patients were treated with RVD induction for three 21-day cycles and randomized 1:1 at inclusion into one of the two mobilization arms CY 2 g/m(2) + G-CSF [arm A] vs. G-CSF alone [arm B]. Plerixafor was given according to a specific algorithm if needed. Sixty-nine patients who received mobilization followed by blood graft collection were included in the cost analysis. The median total costs of the mobilization phase were significantly higher in arm A than in arm B (3855 € vs. 772 €, p ≤ 0.001). The cumulative median cost of the mobilization and collection phases was significantly lower in arm B than in arm A (8524 € vs. 11,622 €, p = 0.012). There was no significant difference between the arms in the total median costs of ASCT (n = 59) (34,997 € in arm A vs. 31,981 € in arm B, p = 0.118). Mobilization with G-CSF alone seems to be a preferable mobilization method for MM patients in terms of mobilization and apheresis costs. In addition, it requires less hospital resource utilization.

The fat mass and obesity-associated (FTO) gene variant rs9939609 predicts long-term incidence of cardiovascular disease and related death independent of the traditional risk factors.

OBJECTIVE AND METHODS: The impact of the rs9939609 FTO variant on cardiovascular events was investigated in the 19-year follow-up of subjects recruited to the OPERA study. RESULTS: A total of 212 cardiovascular disease (CVD) and 152 coronary heart disease (CHD) events or deaths occurred during follow-up. The logistic regression analysis revealed that among the AA genotype the incidence of CHD (OR 1.905; 95% CI 1.250-2.903, p = 0.001) and CVD (OR 1.849; 1.265-2.702, p = 0.003) events or death was significantly higher when adjusted for age, sex, and study group. After further adjustment with BMI, smoking status, systolic blood pressure, and low-density lipoprotein cholesterol, the higher incidence of CHD and CVD events or death among subjects with the AA genotype remained significant (OR 1.895; p = 0.002 and p = 0.004, respectively). In Cox regression analysis, the AA genotype displayed a higher rate of CVD and CHD death when the model was adjusted for sex, age, and study group (p = 0.006 and p = 0.046). FTO rs9939609 AA genotype improved the C-index of the final predictive model from 0.709 to 0.715. In reclassification analyses, the integrated discrimination index was significant 0.011 (p = 0.010). CONCLUSION: The AA genotype of FTO rs9939609 seems to be associated with a higher risk of CVD, and this phenomenon seems to be independent of the traditional risk factors for atherosclerosis.

TIM-family molecules in embryonic hematopoiesis: fetal liver TIM-4(lo) cells have myeloid potential.

Trans-membrane (or T cell) immunoglobulin and mucin (TIM) molecules are known regulators of immune response whose function in hematopoiesis is unknown. Earlier, we found that tim-1 and tim-4 are expressed by CD45(+) cells in the para-aortic region of chicken embryo. Because the para-aortic region is a known site for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) differentiation and expansion, we hypothesize that TIM molecules have a role in hematopoiesis. To study this role further, we analyzed TIM expression more precisely in chicken para-aortic region and mouse fetal liver hematopoietic cells. Additionally, we examined the hematopoietic potential of TIM-4(+) mouse fetal liver cells with a colony-forming assay. tim-1 gene expression was detected in chicken and mouse embryos in the aorta-gonads-mesonephros-region at the time of HSC emergence, whereas tim-3 mRNA was widely expressed in different tissues. tim-4 expression was restricted to fetal liver CD45(+)F4/80(+) cells. Moreover, two TIM-4(+) populations were distinguished: F4/80(hi)TIM-4(hi) and F4/80(lo)TIM-4(lo). F4/80(hi)TIM-4(hi) cells had no hematopoietic potential and were morphologically similar to mature macrophages, suggesting that they are yolk sac-derived macrophages. Instead, many of the F4/80(lo)TIM-4(lo) cells were c-kit(+) and Sca-1(+) and had primitive morphology and multilineage colony-forming ability. In addition, F4/80(lo)TIM-4(lo) cells included a considerable population expressing ER-MP12, a known marker for macrophage colony-forming cells and other myeloid progenitors. We conclude that TIM molecules are expressed in embryonic hematopoietic tissues in chicken and mouse and that in fetal liver, TIM-4 is expressed by myeloid progenitor cells.