NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer-specific survival in patients with aggressive B-cell lymphomas.

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis.

Higher incidence of the SNP Met 788 Ile in the coding region of A20 in diffuse large B cell lymphomas.

Genetic alterations causing constitutive activation of the nuclear factor kappa B (NF-κB) signaling pathway has been associated with the development of lymphomas. A20 (TNFAIP3) is a key regulator of NF-κB signaling. Its suppressor functions are often inactivated by deletions and/or mutations in various hematologic malignancies. Since we recently found the rs143002189 polymorphism in the A20 loci in our multiple myeloma samples, we further investigated this polymorphism in different lymphoid neoplasias. For this purpose, we tested 479 cases of the most common B cell malignancies for the presence of the rs143002189 polymorphism. We found a significant higher occurrence of the rs143002189 polymorphism in diffuse large B cell lymphoma (DLBCL) compared to non-neoplastic controls and other types of B cell malignancies. Furthermore, structure analyses of the mutated A20 protein led to the assumption that the new steric interaction within the protein is responsible for a reduced or inactivated A20 protein. Our data indicates that in a significant fraction of patients, rs143002189 might contribute to the development of DLBCL.

A modified scoring of the NCCN-IPI is more accurate in the elderly and is improved by albumin and ß2 -microglobulin.

The International Prognostic Index (IPI) has been used for decades in diffuse large B-cell lymphoma (DLBCL). A retrospective cancer registry analysis in North America showed significantly improved results when an enhanced IPI, the National Comprehensive Cancer Network (NCCN)-IPI was applied. This novel score puts more weight on age and high levels of lactate dehydrogenase (LDH). Nevertheless, it remains unclear if these results can be extrapolated to the general population. This retrospective bi-centre analysis included 499 unselected DLBCL patients who were treated with rituximab and anthracycline-based chemoimmunotherapy between 2004 and 2013. In our cohort, the NCCN-IPI was more accurate in identifying patients at low or high risk, despite older age, and more patients with increased LDH. Nevertheless, a modified scoring of the risk factors was required to more accurately identify elderly patients with a very favourable diagnosis, suggesting an impaired value of the original NCCN-IPI in the elderly. Serum ß2 -microglobulin and albumin were retained as independent prognostic factors for survival in a multivariate analysis. Our data confirm, for the first time, the superior prognostic power of the NCCN-IPI in an unselected, middle-European cohort. We furthermore propose a modified NCCN-IPI for more accurate prognostication in the elderly. Albumin and ß2 -microglobulin levels are likely to add significant information to the NCCN-IPI.

Independent Prognostic Value of Serum Markers in Diffuse Large B-Cell Lymphoma in the Era of the NCCN-IPI.

BACKGROUND: Several serum parameters have been evaluated for adding prognostic value to clinical scoring systems in diffuse large B-cell lymphoma (DLBCL), but none of the reports used multivariate testing of more than one parameter at a time. The goal of this study was to validate widely available serum parameters for their independent prognostic impact in the era of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) score to determine which were the most useful. PATIENTS AND METHODS: This retrospective bicenter analysis includes 515 unselected patients with DLBCL who were treated with rituximab and anthracycline-based chemoimmunotherapy between 2004 and January 2014. RESULTS: Anemia, high C-reactive protein, and high bilirubin levels had an independent prognostic value for survival in multivariate analyses in addition to the NCCN-IPI, whereas neutrophil-to-lymphocyte ratio, high gamma-glutamyl transferase levels, and platelets-to-lymphocyte ratio did not. CONCLUSIONS: In our cohort, we describe the most promising markers to improve the NCCN-IPI. Anemia and high C-reactive protein levels retain their power in multivariate testing even in the era of the NCCN-IPI. The negative role of high bilirubin levels may be associated as a marker of liver function. Further studies are warranted to incorporate these markers into prognostic models and define their role opposite novel molecular markers.

The significance of pretreatment anemia in the era of R-IPI and NCCN-IPI prognostic risk assessment tools: a dual-center study in diffuse large B-cell lymphoma patients.

BACKGROUND: Anemia is frequently identified at the time of diagnosis in patients with diffuse large B-cell lymphoma (DLBCL); however, studies addressing the prognostic significance of this important clinical parameter are lacking. METHODS: In this dual-center study of patients with DLBCL (n = 556) treated with rituximab-containing regimens, we evaluated the prognostic relevance of anemia at diagnosis in a training set (n = 211) and validated our findings in a second independent patient cohort (n = 345). Using Kaplan-Meier curves as well as univariate and multivariate Cox regression models, we analyzed the impact of anemia on 5-year overall survival (OS) and 5-year disease-free survival (DFS) alongside established prognostic indicators including age, tumor stage, the revised International Prognostic Index (R-IPI), and the recently published NCCN-IPI. The influence of anemia on the predictive accuracy of IPI, R-IPI, and NCCN-IPI prognosis scores was subsequently determined using the Harrell's concordance index. RESULTS: Anemia was an independent predictor of impaired OS and DFS at 5 years in both DLBCL patient cohorts (P < 0.001, log-rank test). In multivariate analysis, hemoglobin level was also a strong and independent prognostic indicator in patients stratified according to R-IPI or NCCN-IPI score. In survival analysis, the estimated concordance index, using IPI, R-IPI, and NCCN-IPI stratification measures (0.69, 0.64, and 0.70, respectively), improved to 0.70, 0.68, and 0.73, respectively, when anemia was also considered. CONCLUSION: In this study, we have demonstrated that anemia at the time of diagnosis is an independent predictor of impaired clinical outcome in DLBCL. Furthermore, consideration of hemoglobin levels may improve the accuracy of recently established prognostic tools in lymphoma. Our data encourage further evaluation of the prognostic utility of this readily accessible biological parameter in prospective clinical trials.

Management of gastric mucosa-associated lymphoid tissue lymphoma.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) type represent 7-8% of all B cell lymphomas and up to 50% of primary gastric lymphomas and can arise at any extranodal site. The most common manifestation is the stomach, which is almost invariably associated with a chronic Helicobacter pylori infection. The diagnosis is based on the histopathological evaluation of multiple gastric biopsies in accordance with the current WHO classification. The mainstay of therapy is H. pylori eradication, which must be delivered to all gastric MALT lymphoma patients, independent of stage. In patients who do not achieve lymphoma regression following antibiotic therapy, irradiation and/or systemic oncological therapies should be applied, depending on the stage of the disease. Radiotherapy might be the preferred option for localized stage. However, in the presence of disseminated or advanced disease, chemotherapy and/or immunotherapy with the anti-CD 20 antibody rituximab is the treatment of choice, but no standard chemotherapy has been defined so far. Gastric MALT lymphomas have a limited tendency to distant spreading and to histological transformation and thus MALT lymphoma usually has a favorable outcome, with an overall survival rate at 5 years of more than 85%.

miR-199a and miR-497 Are Associated with Better Overall Survival due to Increased Chemosensitivity in Diffuse Large B-Cell Lymphoma Patients.

Micro-RNAs (miRNAs) are short non-coding single-stranded RNA molecules regulating gene expression at the post-transcriptional level. miRNAs are involved in cell development, differentiation, apoptosis, and proliferation. miRNAs can either function as tumor suppressor genes or oncogenes in various important pathways. The expression of specific miRNAs has been identified to correlate with tumor prognosis. For miRNA expression analysis real-time PCR on 81 samples was performed, including 63 diffuse large B-cell lymphoma (DLBCL, 15 of germinal center B-cell like subtype, 17 non germinal center B-cell, 23 transformed, and eight unclassified) and 18 controls, including nine peripheral B-cells, 5 germinal-center B-cells, four lymphadenitis samples, and 4 lymphoma cell lines (RI-1, SUDHL4, Karpas, U2932). Expression levels of a panel of 11 miRNAs that have been previously involved in other types of cancer (miR-15b_2, miR-16_1*, miR-16_2, miR-16_2*, miR-27a, miR-27a*, miR-98-1, miR-103a, miR-185, miR-199a, and miR-497) were measured and correlated with clinical data. Furthermore, cell lines, lacking miR-199a and miR-497 expression, were electroporated with the two respective miRNAs and treated with standard immunochemotherapy routinely used in patients with DLBCL, followed by functional analyses including cell count and apoptosis assays. Seven miRNAs (miR-16_1*, miR-16_2*, miR-27a, miR-103, miR-185, miR-199, and miR-497) were statistically significantly up-regulated in DLBCL compared to normal germinal cells. However, high expression of miR-497 or miR-199a was associated with better overall survival (p = 0.042 and p = 0.007). Overexpression of miR-199a and miR-497 led to a statistically significant decrease in viable cells in a dose-dependent fashion after exposure to rituximab and various chemotherapeutics relevant in multi-agent lymphoma therapy. Our data indicate that elevated miR-199a and miR-497 levels are associated with improved survival in aggressive lymphoma patients most likely by modifying drug sensitivity to immunochemotherapy. This functional impairment may serve as a potential novel therapeutic target in future treatment of patients with DLBCL.

Serum and urine galactomannan testing for screening in patients with hematological malignancies.

Testing for serum galactomannan (GM) has been established as an important method for diagnosing invasive aspergillosis (IA); however, limited data exist regarding the application of urine GM testing. The objective of this study was to evaluate the performance of GM screening of urine specimens and to compare results with serum GM. The study was performed between July 2012 and March 2013 in adult patients with underlying hematological malignancies who were hospitalized at the Medical University of Graz, Austria. Serum and urine screening samples were collected and tested twice weekly (always on the same day). In total, 242 serum samples and a similar number of urine samples were collected from 75 patients. A total of 21/242 (8.7%) serum samples from 13 patients were GM positive. Sensitivity, specificity, positive predictive value, and negative predictive value using a 0.1 optical density index cutoff for urine samples (compared with same-day serum results) were as follows: 47.6%, 86%, 24.4%, and 94.5%, respectively. In 8/10 patients with probable IA, at least one positive GM result was found with this cutoff. After calculating clinical performance of the urine GM test, we found that sensitivity increased to 71.4% and specificity to 88.2%. Spearman-Rho correlation analysis revealed a significant positive correlation between serum and urine samples (P < 0.001; ρ = 0.252). In conclusion, GM detection in urine might be a promising method for IA screening. However, further studies are needed.

Potential factors for inadequate voriconazole plasma concentrations in intensive care unit patients and patients with hematological malignancies.

Voriconazole plasma concentrations (VPCs) vary widely, and concentrations outside the therapeutic range are associated with either worse outcome in invasive aspergillosis (IA) or increased toxicity. The primary goal of this cohort study conducted in a real-life setting was to identify potential factors associated with inadequate VPCs in ICU patients and patients with hematological malignancies. Within a period of 12 months, trough VPCs were obtained and analyzed with high-performance liquid chromatography, and the adequate range was defined as 1.5 to 5.5 mg/liter. VPCs of <1.5 mg/liter were defined as low, whereas VPCs of >5.5 mg/liter were defined as potentially toxic. A total of 221 trough VPCs were obtained in 61 patients receiving voriconazole, and 124/221 VPCs (56%) were found to be low. Multivariate analysis revealed that low VPCs were significantly associated with clinical failure of voriconazole, prophylactic use, younger age, underlying hematological malignancy, concomitant proton pump inhibitor (PPI) (pantoprazole was used in 88% of the patients), and absence of side effects. Low VPCs remained an independent predictor of clinical failure of voriconazole. The defined adequate range was reached in 79/221 (36%) VPCs. In 18 samples (8%), potentially toxic levels were measured. Multivariate analysis revealed higher body mass index (BMI), absence of hematological malignancy, therapeutic application, and diarrhea as factors associated with potentially toxic VPCs. Neurotoxic adverse events occurred in six patients and were mostly associated with VPCs in the upper quartile of our defined adequate range. In conclusion, potential factors like younger age, prophylaxis, underlying hematological malignancy, BMI, and concomitant PPI should be considered within the algorithm of voriconazole treatment.

Microbiological screening for earlier detection of central venous catheter-related bloodstream infections.

BACKGROUND: Catheter-related bloodstream infections (CRBSIs) are currently detected with a reactive diagnostic policy, that is, application of tests to patients with clinically suspected CRBSI. The aim of our study was to evaluate whether CRBSIs could be anticipated in an earlier stage by microbiological screening using peptide nucleic acid fluorescence in situ hybridization (PNA FISH) with universal hybridization probes or acridine-orange leucocyte cytospin (AOLC) tests in haemodialysis and haematological patients with CVCs in situ compared with routine test. MATERIALS AND METHODS: Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) and AOLC tests using blood samples from both CVC lines in patients undergoing haemodialysis were performed three times a week and from one CVC line in haematological patients were performed daily. Results were compared with those obtained from routinely performed CRBSI diagnostic tests. RESULTS: One hundred fifteen patients with 139 catheter periods were investigated. The mean observation time per catheter period was 25 days (IQR 13.5-43.5), resulting in 5615 CVC days with a total of 4839 tested blood samples. Five CRBSI cases were detected by routine measures resulting in a CRBSI rate of 0.9/1000 catheter days. Four of five CRBSIs could be anticipated by positive PNA FISH and AOLC tests 2-8 days before the diagnosis was established with routine measures. CONCLUSIONS: The proactive anticipative strategy using microscopic examination of CVC blood samples to anticipate CRBSI in an earlier stage compared with routine measures is a new diagnostic approach in patients with CVCs and a high risk of developing CRBSI.

Anemia management in cancer patients with chronic kidney disease.

Anemia is a common complication of cancer and chronic kidney disease (CKD) associated with decreased physical performance as well as poor prognosis for life expectancy. Renal and cancer-induced anemia share common features regarding pathogenesis and therapeutic strategies. It is typically treated with iron substitution, erythropoiesis-stimulating agents (ESA) and in refractory cases with red blood cell transfusions. However, studies of the past few years unveiled numerous setbacks in the use of ESAs. These included a higher risk of cerebrovascular events and increased mortality without the improvement of cardiovascular outcomes in patients with CKD. Moreover, particularly negative results were observed in patients with previous cancer history under ESA therapy. These unfavorable findings have forced the clinicians to reevaluate the management of renal anemia. This led to decrease of ESA usage, while iron substitution and alternative therapeutic options gained more significance. Iron supplementation is also accompanied with certain risks ranging from gastrointestinal complications to severe allergic reactions and increased rate of infections. Furthermore, the evaluation of the long-term safety of excessive iron therapy is still lacking, especially in CKD patients with cancer. In the absence of these clinical studies, this review aims to summarize the currently available therapeutic strategies in anemia management of CKD patients with concomitant cancer.

The clinical significance of fibrinogen plasma levels in patients with diffuse large B cell lymphoma.

BACKGROUND: Fibrinogen plays a crucial role in the pathophysiology of tumour cell growth, invasion and metastasis. The aim of this study was to evaluate the prognostic significance of pretreatment plasma fibrinogen levels in patients with diffuse large B cell lymphoma (DLBCL) METHODS: Data from 372 patients with DLBCL, diagnosed and treated between 2004 and 2013 at two Austrian centres, were evaluated retrospectively. The prognostic influences of plasma fibrinogen levels and other factors, including age, tumour stage and the National Comprehensive Cancer Network-International Prognostic Index, on 5-year overall survival (OS) and 5-year disease-free survival (DFS) were studied using Kaplan-Meier curves as well as univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis revealed that a high fibrinogen plasma level is associated with decreased 5-year OS and 5-year DFS in patients with DLBCL (p<0.001, log-rank test). Furthermore, in multivariate analysis, elevated serum fibrinogen was found to be an independent marker of poor clinical outcome: 5-year OS (HR=1.69, 95% CI 1.06 to 2.72, p=0.029) and 5-year DFS (HR=1.68, 95% CI 1.08 to 2.61, p=0.021). CONCLUSIONS: In the current study, we demonstrate that high plasma fibrinogen levels at diagnosis predict poor outcome in patients with DLBCL. TRIAL REGISTRATION NUMBER: 25-434 ex 12713 and 415-EP/73/127-2012.

Molecular Pathogenesis of MALT Lymphoma.

Approximately 8% of all non-Hodgkin lymphomas are extranodal marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT), also known as MALT lymphoma, which was first described in 1983 by Isaacson and Wright. MALT lymphomas arise at a wide range of different extranodal sites, with the highest frequency in the stomach, followed by lung, ocular adnexa, and thyroid, and with a low percentage in the small intestine. Interestingly, at least 3 different, apparently site-specific, chromosomal translocations and missense and frameshift mutations, all pathway-related genes affecting the NF-κB signal, have been implicated in the development and progression of MALT lymphoma. However, these genetic abnormalities alone are not sufficient for malignant transformation. There is now increasing evidence suggesting that the oncogenic product of translocation cooperates with immunological stimulation in oncogenesis, that is, the association with chronic bacterial infection or autoaggressive process. This review mainly discusses MALT lymphomas in terms of their genetic aberration and association with chronic infections and summarizes recent advances in their molecular pathogenesis.

The nuclear orphan receptor NR4A1 and NR4A3 as tumor suppressors in hematologic neoplasms.

NR4A1 (Nur77) belongs together with NR4A2 (Nurr1) and NR4A3 (NOR-1) to the nuclear orphan receptors of the NR4A-family. Their activation is generally short lived, the cellular outcome is a stimulus- and cell context-dependent differential activation of NR4A target genes that regulate cell cycle, apoptosis, inflammation, atherogenesis, metabolism, DNA repair and tumorigenesis. NR4A1 and NR4A3 were identified to function as tumor suppressors in acute myeloid leukemia (AML). Deletion of both nuclear receptors led to rapid development of AML in mice. Loss of NR4A1 and NR4A3 was a common feature in human AML patients. Additionally, NR4A1 and NR4A3 hypoallelic mice - mice with a reduced NR4A1 and NR4A3 expression - develop a chronic myeloid malignancy that recapitulates the pathological features of myelodysplastic/ myeloproliferative neoplasms with progression to AML in rare cases. Recently, a reduced NR4A1 and NR4A3 expression was described in aggressive lymphomas and low NR4A1 expression was associated with poor overall survival. Overexpression of NR4A1 in aggressive lymphoma cells led to induction of apoptosis and abrogated tumor growth in a xenograft mouse model. Recently, it was shown that NR4A inducing agents or NR4A agonist possess/induce apoptotic effects in AML and lymphoma cells. Due to this fact and the growing number of NR4A1 and NR4A3 inducing agents and NR4A agonists, both receptors represent new targets for anti tumor therapy.

Frequent down regulation of the tumor suppressor gene a20 in multiple myeloma.

Multiple myeloma (MM) is a malignant clonal expansion of plasma cells in the bone marrow and belongs to the mature B-cell neoplams. The pathogenesis of MM is associated with constitutive NF-κB activation. However, genetic alterations causing constitutive NF-κB activation are still incompletely understood. Since A20 (TNFAIP3) is a suppressor of the NF-κB pathway and is frequently inactivated in various lymphoid malignancies, we investigated the genetic and epigenetic properties of A20 in MM. In total, of 46 patient specimens analyzed, 3 single base pair exchanges, 2 synonymous mutations and one missense mutation were detected by direct sequencing. Gene copy number analysis revealed a reduced A20 gene copy number in 8 of 45 (17.7%) patients. Furthermore, immunohistochemical staining confirmed that A20 expression correlates with the reduction of A20 gene copy number. These data suggest that A20 contributes to tumor formation in a significant fraction of myeloma patients.

Examination of survivin expression in 50 chordoma specimens--A histological and in vitro study.

Chordomas mainly arise along the axial skeleton and are characterized by their slow but destructive growth. Prognosis and quality of life are poor because treatment options are mainly limited to surgery and radiotherapy. Survivin, a member of the apoptosis inhibitor protein family, functions as a key regulator of mitosis and programmed cell death, and is overexpressed in many tumor types. The aim of this study was to determine the role of survivin in chordomas. Survivin expression was investigated in 50 chordoma samples and three chordoma cell lines using immunohistochemistry. The intensity of immunostaining was evaluated in regard to the development of recurrences. The immunohistochemical results were correlated with clinical parameters like gender, age, tumor size, and location and were performed in primary chordomas as well as in recurrent lesions. Furthermore, survivin knockdown experiments on chordoma cell lines were performed. YM155 decreased the growth behavior of chordoma cells dose- and time dependently. Transient knockdown of survivin led to a G2/M arrest, decreased proliferation, consistently induced an increase of polyploidy and morphological changes, and induced apoptosis. The resultant data from this study suggest that survivin plays a cell cycle-progressive role in chordomas. Hence, regulation of survivin by YM155 is a promising new target for the development of new therapeutic drugs.

MicroRNAs in diffuse large B-cell lymphoma: implications for pathogenesis, diagnosis, prognosis and therapy.

Diffuse large B-cell lymphoma (DLBCL) is the most commonly-occurring type of non-Hodgkin lymphoma and is considered a curable disease in at least 50% of patients. Considering that the disease represents a heterogeneous group of tumors, recent efforts using gene expression profiling have identified two subgroups, with significantly different response rates to standard immunochemotherapy. Nevertheless, multiple factors in the pathogenesis of this disease remain unclear and continue to be the focus of further research. MicroRNAs are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. The role of microRNAs in cancer initiation and progression has been demonstrated in multiple types of solid cancers and hematological malignancies such as lymphomas. MicroRNAs also have diagnostic potential, and therapeutic of microRNAs targeting is actively pursued. This review provides an overview on the role of microRNAs in the diagnosis of diffuse large B-cell lymphoma, their role in molecular pathogenesis and hence their prospective role in implementing novel future treatment options.

Safety and tolerability of intrathecal liposomal cytarabine as central nervous system prophylaxis in patients with acute lymphoblastic leukemia.

Central nervous system recurrence in acute lymphoblastic leukemia (ALL) occurs in up to 15% of patients and is frequently associated with poor outcome. The purpose of our study was to evaluate the efficacy and safety of a slow-release liposomal formulation of cytarabine for intrathecal (IT) meningeal prophylaxis in patients suffering from ALL. Forty patients aged 20-77 years (median 36) were preventively treated with a total of 96 (range 1-6) single doses containing 50 mg of liposomal cytarabine on a compassionate use basis. After a median observation period of 23 months (range 2-118) only two patients experienced a combined medullary-leptomeningeal disease recurrence after primary diagnosis. Except for headache grade 2 in two patients, no specific toxicity attributable to IT liposomal cytarabine application was noted. Long-term neurological side effects were not observed. IT liposomal cytarabine therapy with concomitant dexamethasone appears to be feasible and well tolerated.

Evaluation of four molecular assays for detection of pandemic influenza A (H1N1) 2009 virus in the routine diagnostic laboratory.

BACKGROUND: Detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA have gained significance because of their widespread community transmission. OBJECTIVE: To study the accuracy and the performance of four molecular assays for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA in the routine diagnostic laboratory. STUDY DESIGN: The accuracy of the molecular assays was determined with reference material. For evaluation of the performance, 104 clinical specimens were studied. Sample preparation was done on a fully automated extraction instrument. For amplification and detection of influenza RNA, all molecular assays evaluated were based on real-time PCR. RESULTS: When the accuracy was tested, the majority of assays yielded results as expected. When clinical samples were analyzed, 94 samples gave concordant results with all assays. One of the assays showed one false-negative result and another assay 10 false-negatives. CONCLUSION: The majority of assays evaluated in this study proved suitable for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA. All assays are easy to handle and provide results rapidly.