Marie C. Béné

Clinical Utility of Microarray-Based Gene Expression Profiling in the Diagnosis and Subclassification of Leukemia: Report From the International Microarray Innovations in Leukemia Study Group

PURPOSE The Microarray Innovations in Leukemia study assessed the clinical utility of gene expression profiling as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies. METHODS The investigation was performed in 11 laboratories across three continents and included 3,334 patients. An exploratory retrospective stage I study was designed for biomarker discovery and generated whole-genome expression profiles from 2,143 patients with leukemias and myelodysplastic syndromes. The gene expression profiling-based diagnostic accuracy was further validated in a prospective second study stage of an independent cohort of 1,191 patients. RESULTS On the basis of 2,096 samples, the stage I study achieved 92.2% classification accuracy for all 18 distinct classes investigated (median specificity of 99.7%). In a second cohort of 1,152 prospectively collected patients, a classification scheme reached 95.6% median sensitivity and 99.8% median specificity for 14 standard subtypes of acute leukemia (eight acute lymphoblastic leukemia and six acute myeloid leukemia classes, n = 693). In 29 (57%) of 51 discrepant cases, the microarray results had outperformed routine diagnostic methods. CONCLUSION Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to todays state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

Plasma Level of a Triggering Receptor Expressed on Myeloid Cells-1: Its Diagnostic Accuracy in Patients with Suspected Sepsis

Context Exposure to bacteria upregulates an immunoglobulin-like molecule on phagocytes called the triggering receptor expressed on myeloid cells-1 (TREM-1). Can TREM-1 help distinguish sepsis from the systemic inflammatory response syndrome (SIRS) without infection? Contribution In this prospective study, 76 critically ill adults with suspected infection had plasma TREM-1 levels measured with a rapid immunoblot technique. Thirty-eight percent of the adults had SIRS, and 62% had sepsis. A TREM-1 level higher than 60 ng/mL increased the odds of sepsis by 8.6 (95% CI, 3.8 to 21.5). Implications Levels of TREM-1 may help diagnose bacterial sepsis in critically ill patients. The Editors Sepsis is a common cause of morbidity and death in intensive care units (1). Clinical and laboratory signs of systemic inflammation, including changes in body temperature, tachycardia, or leukocytosis, are neither sensitive nor specific enough for the diagnosis of sepsis. These signs can also be misleading because critically ill patients often present with the systemic inflammatory response syndrome but no infection (2-4). This issue is of paramount importance, since therapy and outcome differ greatly between patients with and those without sepsis. Moreover, the widespread use of antibiotics for all such patients is likely to increase antibiotic resistance, toxicity, and costs (5). Thus, there is an as-yet-unsatisfied need for clinical or laboratory tools allowing clinicians to distinguish between the systemic inflammatory response syndrome and sepsis. Among the potentially useful markers of sepsis, procalcitonin has been suggested as the most promising (6-8). However, several investigators have questioned the diagnostic and prognostic accuracy of routine procalcitonin measurements, reporting inconsistent and variable results depending on the severity of illness and infection in the patient sample studied (9-11). The triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily, and its expression is upregulated on phagocytic cells in the presence of bacteria or fungi (12). Several experiments by Bouchon and colleagues (13) showed that TREM-1 mediates the acute inflammatory response to microbial products. Human tissues infected with bacteria are infiltrated with neutrophils and macrophages that express high levels of TREM-1. Conversely, TREM-1 is only weakly expressed in samples from patients with noninfectious inflammatory disorders (13). In addition, TREM-1 is shed from the membrane of activated phagocytes and can be found in a soluble form in body fluids. The presence of a soluble form of TREM-1 in samples of bronchoalveolar lavage fluid from mechanically ventilated patients has been shown to be a good indicator of infectious pneumonia (14). In this study, we prospectively investigated the diagnostic value of an assay measuring the plasma level of soluble TREM-1 in distinguishing sepsis from severe systemic noninfectious inflammation among newly admitted critically ill patients with suspected infection. Methods Study Sample All consecutive patients who were newly hospitalized in the medical intensive care unit of a teaching hospital in France between July and September 2003 were prospectively enrolled in the study if they had clinically suspected infection and fulfilled at least 2 criteria of the systemic inflammatory response syndrome (Appendix Table 1) (15). Clinically suspected infection was defined as an explicit statement by the attending physician indicating suspicion of an ongoing infection. In all enrolled patients, diagnostic work-up was performed to identify or rule out infection and antimicrobial therapy was prescribed. Patients were not enrolled if they were older than 80 years of age or were immunocompromised because of treatment with corticosteroids, receipt of bone marrow or organ transplants, leukopenia (leukocyte count < 1.0 109 cells/L) or neutropenia (polymorphonuclear granulocyte count < 0.5 109 cells/L), a hematologic malignant condition, or AIDS. Patients who died or were discharged early (within 12 hours after admission) or presented with complete absence of antimicrobial treatment were also excluded. Patients originated from the emergency department, the general wards, or the operating room. The hospitals institutional review board approved the study, and we obtained informed consent from patients or their relatives before inclusion. Data Collection At admission to the intensive care unit, we recorded the following items for each patient: age; sex; severity of the underlying medical condition, stratified according to the criteria of McCabe and Jackson (16); Simplified Acute Physiology Score II (range, 0 to 194, with a higher score indicating worse condition) (17); Sepsis-related Organ Failure Assessment score (range, 0 to 24; scores for each organ system [respiration, coagulation, liver, cardiovascular, central nervous system, and kidney] ranged from 0 [normal] to 4 [most abnormal]) (18); reason for admission to the intensive care unit; principal diagnosis; vital signs; respiratory variables; and results of routine blood tests and microbiological culture results. Survival or death in the intensive care unit was assessed during a follow-up period as long as 28 days. The attending physician prescribed microbiological tests and antimicrobial therapy according to the usual practice of the intensive care unit, without interference by the research team. Two intensivists retrospectively reviewed all medical records pertaining to each patient and independently classified the diagnosis as the systemic inflammatory response syndrome (that is, no infection), sepsis, severe sepsis, or septic shock at the time of admission, according to established consensus definitions (Appendix Table 1) (15). Agreement about the diagnosis was achieved in all cases. Both intensivists were blinded to plasma soluble TREM-1 values. Measurements of Plasma Levels of Procalcitonin and Soluble TREM-1 Within 12 hours after admission and study enrollment, 5 mL of whole heparinized blood was drawn through an arterial line for measurement of procalcitonin and soluble TREM-1 levels. Plasma was collected by centrifugation at 4 C, separated into aliquots, and stored at 80 C until the day of assay. Plasma procalcitonin concentrations were measured by using an immunoassay with a sandwich technique and a chemiluminescent detection system, according to the manufacturers protocol (LUMITest, Brahms Diagnostica, Berlin, Germany). Plasma soluble TREM-1 levels were assessed as described elsewhere (14). Briefly, 100 L of each plasma sample was dotted on a nitrocellulose membrane, dried, and overcoated in phosphate-buffered saline solution supplemented with 3% bovine serum albumin. The nitrocellulose sheet was then incubated for 60 minutes in the presence of monoclonal antiTREM-1 antibody. After thorough rinsing, the sheet was further incubated for 60 minutes with diluted (1:1000) goat anti-mouse immunoglobulins (Dako, Glostrup, Denmark), washed in phosphate-buffered saline solution supplemented with 20% dimethylsulfoxide, and incubated for 30 minutes with diluted (1:1000) horseradish peroxidaseconjugated streptavidin (Bio-Rad, Cergy, France). The enzyme substrate chromogen Opti-4CN (Bio-Rad) was then added, and color developed in proportion to the amount of soluble TREM-1 bound to the membrane. Each sheet also contained calibration samples of a known concentration of soluble TREM-1 (0 to 5000 ng/mL). Colorimetric determination was achieved by using a reflectance scanner and Quantity One quantitation software (Bio-Rad). Soluble TREM-1 concentration was determined in each sample by plotting the optical densities of the samples to the standard curve. All measurements were performed in duplicate, and results were expressed as mean concentration in ng/mL of plasma. The sensitivity of this technique allows the detection of soluble TREM-1 levels as low as 5 ng/mL, and the entire procedure takes less than 3 hours. The coefficient of variation of the assay was lower than 5%. Statistical Analysis Descriptive results of continuous variables were expressed as the mean (SD). Plasma soluble TREM-1 and procalcitonin levels were expressed as the median and range. Variables were tested for their association with the diagnosis by using the Pearson chi-square test for categorical data and the MannWhitney U test for numerical data. The different groups were compared by using the MannWhitney U test (or nonparametric KruskalWallis test when appropriate) for numerical data and the Pearson chi-square test (or the Fisher exact test when appropriate) for categorical data. The relation between soluble TREM-1 level and clinical or biological features was assessed by using the Spearman correlation test. Receiver-operating characteristic curves were constructed to illustrate various cutoff values of soluble TREM-1, procalcitonin, and C-reactive protein. Sensitivity, specificity, and positive and negative likelihood ratios and their confidence intervals were calculated (19) for the cutoff point that represented the best discrimination, as derived from the areas under the receiver-operating characteristic curves. We used StatView software (Abacus Concepts, Berkeley, California) to complete the analysis. A 2-tailed P value less than 0.05 was considered statistically significant. Role of the Funding Source The funding source had no role in the design, conduct, or reporting of the study or the decision to publish the manuscript. Results Characteristics of the Study Sample From July to September 2003, 98 patients were admitted into our intensive care unit with clinical suspicion of infection. Of these, 22 were not included in the study because of early death, immunocompromised state, age older than 80 years, absence of consent (for 2 patients), or protocol violation (for 5 patients who were not enrolled because an attending physician was unaware of

A Soluble Form of the Triggering Receptor Expressed on Myeloid Cells-1 Modulates the Inflammatory Response in Murine Sepsis

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently discovered receptor expressed on the surface of neutrophils and a subset of monocytes. Engagement of TREM-1 has been reported to trigger the synthesis of proinflammatory cytokines in the presence of microbial products. Previously, we have identified a soluble form of TREM-1 (sTREM-1) and observed significant levels in serum samples from septic shock patients but not controls. Here, we investigated its putative role in the modulation of inflammation during sepsis. We observed that sTREM-1 was secreted by monocytes activated in vitro by LPS and in the serum of animals involved in an experimental model of septic shock. Both in vitro and in vivo, a synthetic peptide mimicking a short highly conserved domain of sTREM-1 appeared to attenuate cytokine production by human monocytes and protect septic animals from hyper-responsiveness and death. This peptide seemed to be efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines. These data suggest that in vivo modulation of TREM-1 by sTREM peptide might be a suitable therapeutic tool for the treatment of sepsis.

Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes

This article decribes the results of the first European LeukemiaNet working conference on flow cytometry immunophenotyping in myelodysplastic syndrome. This report is a very comprehensive analysis of the topic, and provides detailed information on what is currently known in the field. See related perspective article on page 1041. The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34+ precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission.

Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia

Not all patients with core binding factor acute myeloid leukemia (CBF-AML) display a good outcome. Modern risk factors include KIT and/or FLT3 gene mutations and minimal residual disease (MRD) levels, but their respective values have never been prospectively assessed. A total of 198 CBF-AML patients were randomized between a reinforced and a standard induction course, followed by 3 high-dose cytarabine consolidation courses. MRD levels were monitored prospectively. Gene mutations were screened at diagnosis. Despite a more rapid MRD decrease after reinforced induction, induction arm did not influence relapse-free survival (RFS) (64% in both arms; P = .91). Higher WBC, KIT, and/or FLT3-ITD/TKD gene mutations, and a less than 3-log MRD reduction after first consolidation, were associated with a higher specific hazard of relapse, but MRD remained the sole prognostic factor in multivariate analysis. At 36 months, cumulative incidence of relapse and RFS were 22% vs 54% (P < .001) and 73% vs 44% (P < .001) in patients who achieved 3-log MRD reduction vs the others. These results suggest that MRD, rather than gene mutations, should be used for future treatment stratifications in CBF-AML patients. This trial was registered at EudraCT as #2006-005163-26 and at www.clinicaltrials.gov as #NCT 00428558.

Influence of visual control, conduction, and central integration on static and dynamic balance in healthy older adults

Aging is associated with decreased balance abilities, resulting in an increased risk of fall. In order to appreciate the visual, somatosensory, and central signals involved in balance control, sophisticated methods of posturography assessment have been developed, using static and dynamic tests, eventually associated with electromyographic measurements. We applied such methods to a population of healthy older adults in order to appreciate the respective importance of each of these sensorial inputs in aging individuals. Posture control parameters were recorded on a force-measuring platform in 41 healthy young (age 28.5 +/- 5.9 years) and 50 older (age 69.8 +/- 5.9 years) adults, using a static test and two dynamic tests performed by all individuals first with eyes open, then with eyes closed. The distance covered by the center of foot pressure, sway area, and anteroposterior oscillations were significantly higher, with eyes open or closed, in older people than in young subjects. Significant differences were noted in dynamic tests with longer latency responses in the group of old people. Dynamic recordings in a sinusoidal test had a more regular pattern when performed eyes open in both groups and evidenced significantly greater instability in old people. These data suggest that vision remains important in maintaining postural control while conduction and central integration become less efficient with age.

Oncogenetics and minimal residual disease are independent outcome predictors in adult patients with acute lymphoblastic leukemia

With intensified pediatric-like therapy and genetic disease dissection, the field of adult acute lymphoblastic leukemia (ALL) has evolved recently. In this new context, we aimed to reassess the value of conventional risk factors with regard to new genetic alterations and early response to therapy, as assessed by immunoglobulin/T-cell receptor minimal residual disease (MRD) levels. The study was performed in 423 younger adults with Philadelphia chromosome-negative ALL in first remission (265 B-cell precursor [BCP] and 158 T-cell ALL), with cumulative incidence of relapse (CIR) as the primary end point. In addition to conventional risk factors, the most frequent currently available genetic alterations were included in the analysis. A higher specific hazard of relapse was independently associated with postinduction MRD level ≥10(-4) and unfavorable genetic characteristics (ie, MLL gene rearrangement or focal IKZF1 gene deletion in BCP-ALL and no NOTCH1/FBXW7 mutation and/or N/K-RAS mutation and/or PTEN gene alteration in T-cell ALL). These 2 factors allowed definition of a new risk classification that is strongly associated with higher CIR and shorter relapse-free and overall survival. These results indicate that genetic abnormalities are important predictors of outcome in adult ALL not fully recapitulated by early response to therapy. Patients included in this study were treated in the multicenter GRAALL-2003 and GRAALL-2005 trials. Both trials were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

ABSTRACT The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1β, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.