Zdravko Mitrović

LightCycler SeptiFast assay as a tool for the rapid diagnosis of sepsis in patients during antimicrobial therapy.

Rapid aetiological diagnosis and early administration of adequate antimicrobial therapy soon after a patient’s arrival at the hospital are crucial for the successful management of sepsis and septic shock (Dombrovskiy et al., 2007; Raghavan & Marik, 2006). Routine aetiological diagnosis of sepsis relies on standard culture methods that take at least 24 h or more to give the initial information. Therefore, quick identification of pathogens causing sepsis within the clinically relevant time frame should be based on state-of-the-art molecular methods that target bacterial and fungal DNA (Abdul-Redha et al., 2007; Breitkopf et al., 2005; Dombrovskiy et al., 2007). In this letter, we describe the clinical utility of the first standardized, CE-certified, multiplex real-time PCR assay for the molecular diagnosis of sepsis that has been approved for in vitro diagnostic use (LightCycler SeptiFast assay; Roche Diagnostics). The aim of our preliminary study was to determine the additional diagnostic value of the LightCycler SeptiFast assay, which enables detection of DNA from 25 human pathogens (Gram-positive and Gram-negative bacteria as well as fungi) in the blood of patients with suspected sepsis even after empirical antimicrobial therapy has been started. The study enrolled 36 patients (a total of 39 samples) with a clinical diagnosis of sepsis that were treated at the University Hospital for Infectious Diseases, Zagreb, and Zagreb University Clinical Center in Croatia. Seventeen patients were hospitalized in the intensive care unit (ICU), nine patients outside the ICU and ten patients in the Department of Haematology following bone marrow or peripheral blood stem cell transplantation (BSCT). All patients enrolled in the study were already receiving empirical antimicrobial therapy at the time of testing. Blood cultures were taken from all patients as well. Thirteen out of 39 (33 %) samples tested positive with the SeptiFast assay for bacterial or fungal DNA (Table 1). Gramnegative bacterial DNA was detected in 11 of 13 samples (Klebsiella pneumoniae/oxytoca n54; Escherichia coli n53; Pseudomonas aeruginosa n54). Gram-positive bacterial DNA (Enterococcus faecium n51; Streptococcus pneumoniae n51) was detected in two patients with polymicrobial sepsis (in combination with K. pneumoniae/ oxytoca in both patients). Aspergillus fumigatus DNA was detected in two patients with fever and pulmonary infiltrates. Additional SeptiFast-positive results (blood culture-negative) were obtained in nine of 36 patients. Four of 39 samples (10.3 %) were negative by the SeptiFast assay but positive by culture and those results were interpreted as false-negatives. Four of 39 samples were positive by both the SeptiFast assay and culture. Twenty-two samples tested negative by both techniques. We observed one discordant result between the SeptiFast assay and culture (Enterobacter cloacae was detected by culture and K. pneumoniae/oxytoca by SeptiFast assay). Incorrect interpretation of Enterobacter aerogenes/cloacae as K. pneumoniae/oxytoca due to the similarities of internal transcribed spacer regions of the target micro-organisms was also described by the manufacturer of the assay (4.2 % error rate in the evaluation study). The SeptiFast assay does not detect Actinobacillus sp. DNA, but this microorganism was detected in one patient by culture only. In the group of 10 haematological patients, SeptiFast results were positive for six of the 10 patients (60 %), whereas blood cultures were positive in only two out of 10 patients (20 %). The declared analytical sensitivity of the SeptiFast assay ranges between 30 and 100 c.f.u. (depending on the micro-organism). The SeptiFast assay provides a rapid identification of the causative microorganism within 6 h (3 h of technician hands-on time) whereas blood cultures usually require 2 days for Gram-staining results and a total of 3 days for the species to be identified. However, the use of the SeptiFast assay is limited to well-established molecular laboratories, requires excellent technical skills and is very expensive compared to culture. Additionally, the SeptiFast assay cannot provide information regarding the antibiotic susceptibility of micro-organisms. We conclude that the SeptiFast assay is a clinically valuable add-on to conventional culture methods for rapid aetiological diagnosis of sepsis in patients where empirical antimicrobial therapy has already been started and pretreatment blood cultures were negative. This assay showed particular sensitivity in haematological patients following BSCT,

Lack of prognostic significance of the germinal-center phenotype in diffuse large B-cell lymphoma patients treated with CHOP-like chemotherapy with and without rituximab

The influence of the germinal-center B-cell (GCB) and the non-GCB phenotypes of diffuse large B-cell lymphoma (DLBCL) on the outcome of 92 patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like chemotherapy, with or without rituximab was determined in this study. The differentiation between the GCB and non-GCB types was arrived at by immunohistochemistry using previously published criteria. Thirty-nine patients had the GCB and 53 had the non-GCB type of DLBCL. Forty-nine patients were treated with rituximab and chemotherapy; 43 were treated with chemotherapy alone. The GCB and non-GCB group did not differ in their international prognostic index factors and score, presence of bulky disease, or frequency of rituximab treatment. Median follow-up of the surviving patients was carried out for 37 months. There was no difference between the GCB and non-GCB groups in both overall response rates (67 vs. 70%, respectively) and estimated rates of 3-year event-free (46 vs. 49%, respectively) and overall (54 vs. 56%, respectively) survival. In addition, no differences of the outcomes were observed between the subgroups treated with or without rituximab. The patients of this study with immunohistochemically determined GCB-type DLBCL did not have an improved prognosis, irrespective of whether they had received rituximab or not.

Biological prognostic markers in diffuse large B-cell lymphoma.

BACKGROUND Multiple novel therapeutic options have emerged in the treatment of non-Hodgkin lymphoma, including monoclonal antibodies and different classes of biological agents. With this increased diagnostic sophistication, novel prognostic markers are needed to stratify patients according to risk factors, particularly those with a mechanistic underpinning, to provide the basis for individually tailored treatment. METHODS Numerous prognostic markers have been proposed in patients with diffuse large B-cell lymphoma (DLBCL), and this review discusses the more studied and the most widely used prognostic markers in DLBCL in the rituximab era. RESULTS Prognostic markers in DLBCL include a range of biomarkers assessed by morphology, immunohistochemistry, and relatively novel molecular methods including gene expression profiling, high-resolution array comparative genomic hybridization, and next-generation sequencing. Most of these methods are not routinely used due to substantial cost, technical complexity, and the requirement for fresh or frozen tissue. CONCLUSIONS Efforts are underway to translate previous microarray findings to platforms that can be readily used in routine clinical practice with high reproducibility, precise measurements, and minimal loss of information. At the present time, there is no consensus on which biological prognostic markers should be routinely assessed in patients with DLBCL, and practices vary widely among different institutions. With more global approaches, the ability to assess biomarkers in the cellular or tumor context may be possible, resulting in a better understanding of their biological and prognostic significance.

CD43 Expression Is an Adverse Prognostic Factor in Diffuse Large B-Cell Lymphoma

BACKGROUND CD43 is a transmembrane glycoprotein expressed in different hematopoietic cells, including some subsets of B lymphocytes. About a quarter of diffuse large B-cell lymphomas (DLBCLs) express CD43, but its prognostic significance is unknown. PATIENTS AND METHODS We analyzed the prognostic effect of immunohistochemically determined CD43 expression in 119 patients with newly diagnosed DLBCL. All were treated with CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone)-like chemotherapy, 57 without and 62 with rituximab. RESULTS A total of 31 DLBCL cases (26%) expressed CD43. Patients with CD43+ and CD43- lymphomas did not differ regarding sex, International Prognostic Index (IPI) factors and score, rituximab treatment, presence of bulky disease, or germinal center subtype. Median follow-up was 45 months. Patients with CD43+ DLBCL had significantly lower complete response rates (59% vs. 80%; P = .019), 2-year event-free survival (EFS) rates (34% vs. 64%; P = .003), and overall survival (OS) rates (45% vs. 76%; P = .002). The prognostic significance of CD43 expression was retained in multivariate analysis (relative risk [RR] 2.04; P = .013 for EFS; RR 2.17; P = .016 for OS). In subgroup analysis, the effect of CD43 expression was significant in patients treated with rituximab and those with low IPI, whereas it was not reached in patients treated without rituximab. The effect was not observed in patients with high IPI. CONCLUSION These results indicate that CD43 expression is an important independent adverse prognostic factor in DLBCL.

Prognostic significance of survivin and caspase-3 immunohistochemical expression in patients with diffuse large B-cell lymphoma treated with rituximab and CHOP.

Survivin is an inhibitor of apoptosis whose expression may be associated with inferior outcome in patients with diffuse large B-cell lymphoma (DLBCL) treated without rituximab. Caspase-3 is the final caspase of the apoptotic cascade and its pattern of expression may also be related to patients’ outcome. In this study we investigated immunohistochemical expression of survivin and caspase-3 (CPP32) in 57 patients with DLBCL treated with rituximab and CHOP (R-CHOP). According to previously published criteria, we separately analyzed correlation of different types of survivin expression with patients’ outcome. Nuclear survivin was expressed in only 26% of cases, cytoplasmic survivin was expressed in 81% of cases while application of immunoreactivity scoring system yielded 58% of survivin positive cases. Caspase-3 was expressed in 77% of cases. There were no significant correlations between any type of survivin expression and response to treatment or survival of the patients. The expression of caspase-3 was also not associated with patients’ outcome. We conclude that survivin and caspase-3 have no significant prognostic significance in patients with DLBCL treated with R-CHOP.

The Degree of Anisocytosis Predicts Survival in Patients with Primary Myelofibrosis

Background: Red cell distribution width (RDW) provides a quantitative measure of anisocytosis and it is associated with the presence of subclinical systemic inflammation and a poor outcome in a variety of diseases when elevated. Anisocytosis is a feature of primary myelofibrosis (PMF) but it’s prognostic role in PMF has not yet been evaluated. Aims: To determine whether anisocytosis bears prognostic significance in patients with PMF and its relation to disease features. Methods: 33 newly-diagnosed patients with PMF were analyzed in this study. Baseline RDW values were obtained in addition to other routine blood analyses (CRP, LDH, complete blood count and iron metabolism parameters) and JAK2 V617F mutational status. Patients were staged according to IPSS prognostic scoring system, liver and spleen size were assessed by palpation. The Mann Whitney U test, the Pearson correlation and the Χ2 test/ the Fisher test were used where appropriate. Survival analyses were performed using methods of Kaplan and Meier, the log-rank test and the Cox regression analysis. All statistical tests were two-sided and P values <0.05 were considered significant. Results: Median RDW was 19.0% (15.2% - 22.5%). RDW correlated significantly with hemoglobin (p=0.005), CRP (p=0.031), spleen size (p=0.036) and IPSS score (p=0.003). Patients with more pronounced anisocytosis had an inferior overall survival (OS) – very-high RDW (≥19.0%) vs. high RDW (15.1% - 18.9%) subgroup, HR 5.37, p=0.002. RDW remained significantly associated with OS (p=0.002) in a multivariate model including IPSS score, hemoglobin level and CRP. Summary/Conclusion: PMF pathogenesis surpasses inflammation as only cause of anisocytosis. A higher degree of anisocytosis is associated with more advanced disease features and a decreased overall survival. RDW encompasses standard prognostic score and may help in the rapid detection of patients with an unfavorable prognosis.

CD13+ anaplastic large cell lymphoma with leukemic presentation and additional chromosomal abnormality

Anaplastic large cell lymphoma (ALCL) is a highly malignant neoplasm characterized by pleomorphic appearance, different immunophenotypes and variable sites of involvement. Expression of myeloid‐associated markers in anaplastic large cell lymphomas may mislead the medical team and result in delay of diagnosis due to unusual phenotype. It is important to diagnose this type of tumors and distinguish it from myeloid neoplasms (extramedullary myeloid cell tumors and histiocytic tumors) since therapy and prognosis are significantly different.

Rituximab with dose-adjusted EPOCH as first-line treatment in patients with highly aggressive diffuse large B-cell lymphoma and autologous stem cell transplantation in selected patients.

Aim To assess the benefit of rituximab with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (R-DA-EPOCH) regimen as a first-line treatment for patients with diffuse large B-cell lymphoma (DLBCL) presenting with unfavorable or aggressive features, and autologous stem cell transplantation (ASCT) as a part of the first-line treatment for selected DLBCL patients with additional aggressive features. Methods We retrospectively analyzed 75 newly diagnosed DLBCL patients with Ki-67+≥80% or International Prognostic Index ≥2 who were treated with R-DA-EPOCH between 2005 and 2015. Of 24 DLBCL patients with additional aggressive features (Ki-67+≥90% or age-adjusted IPI≥2) who were planned to receive consolidation with ASCT, 17 patients underwent the procedure. We determined the overall response rate (ORR), complete remission (CR), partial remission (PR), 5-year overall survival (OS), and progression free survival (PFS) in all DLBCL patients and specifically those planned to receive ASCT. Results All 75 patients included in the analysis started one or more cycles of therapy. The ORR, CR, and PR rates were 80%, 55%, and 25%, respectively. The response was non-evaluable in 10 of 75 patients due to treatment discontinuation. The OS and PFS rates for all 75 patients were 70% and 61%, respectively, and 80% and 79%, respectively, for 24 planned-to-receive-ASCT patients. Age (≤65 vs >65 years) had no prognostic impact on OS and PFS (P = 0.994 and P = 0.827, respectively). Conclusion Our retrospective analysis of one of the largest DLBCL patient cohorts outside the US National Cancer Institute showed that R-DA-EPOCH is a very effective therapeutic option as a first-line treatment of DLBCL patients with unfavorable prognostic features irrespective of their age. ASCT provided additional benefit for DLBCL patients with additional aggressive features.

LightCycler SeptiFast assay as a tool for the rapid diagnosis of sepsis in patients receiving antimicrobial therapy

Rapid aetiological diagnosis and early administration of adequate antimicrobial therapy soon after a patient’s arrival at the hospital are crucial for the successful management of sepsis and septic shock (Dombrovskiy et al., 2007; Raghavan & Marik, 2006). Routine aetiological diagnosis of sepsis relies on standard culture methods that take at least 24 h or more to give the initial information. Therefore, quick identification of pathogens causing sepsis within the clinically relevant time frame should be based on state-of-the-art molecular methods that target bacterial and fungal DNA (Abdul-Redha et al., 2007; Breitkopf et al., 2005; Dombrovskiy et al., 2007). In this letter, we describe the clinical utility of the first standardized, CE-certified, multiplex real-time PCR assay for the molecular diagnosis of sepsis that has been approved for in vitro diagnostic use (LightCycler SeptiFast assay; Roche Diagnostics). The aim of our preliminary study was to determine the additional diagnostic value of the LightCycler SeptiFast assay, which enables detection of DNA from 25 human pathogens (Gram-positive and Gram-negative bacteria as well as fungi) in the blood of patients with suspected sepsis even after empirical antimicrobial therapy has been started. The study enrolled 36 patients (a total of 39 samples) with a clinical diagnosis of sepsis that were treated at the University Hospital for Infectious Diseases, Zagreb, and Zagreb University Clinical Center in Croatia. Seventeen patients were hospitalized in the intensive care unit (ICU), nine patients outside the ICU and ten patients in the Department of Haematology following bone marrow or peripheral blood stem cell transplantation (BSCT). All patients enrolled in the study were already receiving empirical antimicrobial therapy at the time of testing. Blood cultures were taken from all patients as well. Thirteen out of 39 (33 %) samples tested positive with the SeptiFast assay for bacterial or fungal DNA (Table 1). Gramnegative bacterial DNA was detected in 11 of 13 samples (Klebsiella pneumoniae/oxytoca n54; Escherichia coli n53; Pseudomonas aeruginosa n54). Gram-positive bacterial DNA (Enterococcus faecium n51; Streptococcus pneumoniae n51) was detected in two patients with polymicrobial sepsis (in combination with K. pneumoniae/ oxytoca in both patients). Aspergillus fumigatus DNA was detected in two patients with fever and pulmonary infiltrates. Additional SeptiFast-positive results (blood culture-negative) were obtained in nine of 36 patients. Four of 39 samples (10.3 %) were negative by the SeptiFast assay but positive by culture and those results were interpreted as false-negatives. Four of 39 samples were positive by both the SeptiFast assay and culture. Twenty-two samples tested negative by both techniques. We observed one discordant result between the SeptiFast assay and culture (Enterobacter cloacae was detected by culture and K. pneumoniae/oxytoca by SeptiFast assay). Incorrect interpretation of Enterobacter aerogenes/cloacae as K. pneumoniae/oxytoca due to the similarities of internal transcribed spacer regions of the target micro-organisms was also described by the manufacturer of the assay (4.2 % error rate in the evaluation study). The SeptiFast assay does not detect Actinobacillus sp. DNA, but this microorganism was detected in one patient by culture only. In the group of 10 haematological patients, SeptiFast results were positive for six of the 10 patients (60 %), whereas blood cultures were positive in only two out of 10 patients (20 %). The declared analytical sensitivity of the SeptiFast assay ranges between 30 and 100 c.f.u. (depending on the micro-organism). The SeptiFast assay provides a rapid identification of the causative microorganism within 6 h (3 h of technician hands-on time) whereas blood cultures usually require 2 days for Gram-staining results and a total of 3 days for the species to be identified. However, the use of the SeptiFast assay is limited to well-established molecular laboratories, requires excellent technical skills and is very expensive compared to culture. Additionally, the SeptiFast assay cannot provide information regarding the antibiotic susceptibility of micro-organisms. We conclude that the SeptiFast assay is a clinically valuable add-on to conventional culture methods for rapid aetiological diagnosis of sepsis in patients where empirical antimicrobial therapy has already been started and pretreatment blood cultures were negative. This assay showed particular sensitivity in haematological patients following BSCT,