Christian Langer

Continuous Lenalidomide Treatment for Newly Diagnosed Multiple Myeloma

BACKGROUND Lenalidomide has tumoricidal and immunomodulatory activity against multiple myeloma. This double-blind, multicenter, randomized study compared melphalan-prednisone-lenalidomide induction followed by lenalidomide maintenance (MPR-R) with melphalan-prednisone-lenalidomide (MPR) or melphalan-prednisone (MP) followed by placebo in patients 65 years of age or older with newly diagnosed multiple myeloma. METHODS We randomly assigned patients who were ineligible for transplantation to receive MPR-R (nine 4-week cycles of MPR followed by lenalidomide maintenance therapy until a relapse or disease progression occurred [152 patients]) or to receive MPR (153 patients) or MP (154 patients) without maintenance therapy. The primary end point was progression-free survival. RESULTS The median follow-up period was 30 months. The median progression-free survival was significantly longer with MPR-R (31 months) than with MPR (14 months; hazard ratio, 0.49; P<0.001) or MP (13 months; hazard ratio, 0.40; P<0.001). Response rates were superior with MPR-R and MPR (77% and 68%, respectively, vs. 50% with MP; P<0.001 and P=0.002, respectively, for the comparison with MP). The progression-free survival benefit associated with MPR-R was noted in patients 65 to 75 years of age but not in those older than 75 years of age (P=0.001 for treatment-by-age interaction). After induction therapy, a landmark analysis showed a 66% reduction in the rate of progression with MPR-R (hazard ratio for the comparison with MPR, 0.34; P<0.001) that was age-independent. During induction therapy, the most frequent adverse events were hematologic; grade 4 neutropenia was reported in 35%, 32%, and 8% of the patients in the MPR-R, MPR, and MP groups, respectively. The 3-year rate of second primary tumors was 7% with MPR-R, 7% with MPR, and 3% with MP. CONCLUSIONS MPR-R significantly prolonged progression-free survival in patients with newly diagnosed multiple myeloma who were ineligible for transplantation, with the greatest benefit observed in patients 65 to 75 years of age. (Funded by Celgene; MM-015 ClinicalTrials.gov number, NCT00405756.).

MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML). METHODS Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features--that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature. RESULTS Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1beta. CONCLUSIONS A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways.

Prognostic Significance of, and Gene and MicroRNA Expression Signatures Associated With, CEBPA Mutations in Cytogenetically Normal Acute Myeloid Leukemia With High-Risk Molecular Features: A Cancer and Leukemia Group B Study

PURPOSE To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated). PATIENTS AND METHODS One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients. RESULTS CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes. CONCLUSION Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.

High Expression Levels of the ETS-Related Gene, ERG, Predict Adverse Outcome and Improve Molecular Risk-Based Classification of Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To validate ERG overexpression as an adverse predictor and assess its prognostic value in the context of other molecular markers in cytogenetically normal (CN) -acute myeloid leukemia (AML). PATIENTS AND METHODS Seventy-six adult patients with primary CN-AML, younger than 60 years and treated on Cancer and Leukemia Group B (CALGB) trial 19808, were evaluated for ERG expression by quantitative reverse transcriptase polymerase chain reaction. They were then combined with 72 patients enrolled onto CALGB 9621 for analyses that included other molecular markers. RESULTS Similar to patients enrolled onto CALGB 9621, high ERG expressers on CALGB 19808 had fewer complete remissions (CRs; P = .03) and worse event-free survival (EFS; P = .016) than low ERG expressers. In the combined set, high expressers (n = 55) had fewer CRs (P = .004) and shorter EFS (P < .001) than low expressers (n = 93). High ERG predicted failure to achieve CR (P = .004) after adjusting for BAALC expression (P = .04) and age (P = .008), and EFS (P = .004) after adjusting for FLT3 internal tandem duplication (ITD; P < .001). Among patients without FLT3-ITD (FLT3-ITD negative), only high ERG predicted shorter EFS (P = .001). Among NPM1-mutated (NPM1 positive) patients, high ERG predicted shorter EFS (P = .003), after adjusting for FLT3-ITD (P < .001). When all three markers were considered together, in the favorable FLT3-ITD-negative/NPM1-positive subset, high ERG was the only factor predicting shorter EFS (P = .002). CONCLUSION We validated ERG overexpression as an adverse predictor in CN-AML. Moreover, by using ERG expression levels, we improved the previously proposed molecular-risk classification of CN-AML based on the presence or absence of FLT3-ITD and NPM1 mutations, given that we identified subsets with different outcome among FLT3-ITD-negative, NPM1-positive, and FLT3-ITD-negative/NPM1-positive patients.

High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: A Cancer and Leukemia Group B (CALGB) study

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile.

Prognostic Importance of MN1 Transcript Levels, and Biologic Insights From MN1-Associated Gene and MicroRNA Expression Signatures in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in the context of other predictive molecular markers, and to derive MN1 associated gene- and microRNA-expression profiles in cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS MN1 expression was measured in 119 untreated primary CN-AML adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction. Patients were also tested for FLT3, NPM1, CEBPA, and WT1 mutations, MLL partial tandem duplications, and BAALC and ERG expression. Gene- and microRNA-expression profiles were attained by performing genome-wide microarray assays. Patients were intensively treated on two first-line Cancer and Leukemia Group B clinical trials. Results Higher MN1 expression associated with NPM1 wild-type (P < .001), increased BAALC expression (P = .004), and less extramedullary involvement (P = .01). In multivariable analyses, higher MN1 expression associated with a lower complete remission rate (P = .005) after adjustment for WBC; shorter disease-free survival (P = .01) after adjustment for WT1 mutations, FLT3 internal tandem duplications (FLT3-ITD), and high ERG expression; and shorter survival (P = .04) after adjustment for WT1 and NPM1 mutations, FLT3-ITD, and WBC. Gene- and microRNA-expression profiles suggested that high MN1 expressers share features with high BAALC expressers and patients with wild-type NPM1. Higher MN1 expression also appears to be associated with genes and microRNAs that are active in aberrant macrophage/monocytoid function and differentiation. CONCLUSION MN1 expression independently predicts outcome in CN-AML patients. The MN1 gene- and microRNA-expression signatures suggest biologic features that could be exploited as therapeutic targets.

Serum microRNAs as a novel class of biomarkers: a comprehensive review of the literature

Recently, expression patterns of microRNAs in body fluids underscored their potential as noninvasive biomarkers for various diseases. Here we summarize the current works describing microRNAs in blood cells or serum as biomarkers and the applied methodologies of small RNA purification and detection. Future challenges and the required research relating to optimization of working with microRNAs in serum will also be discussed.

Common variation at 3q26.2, 6p21.33, 17p11.2 and 22q13.1 influences multiple myeloma risk

To identify variants for multiple myeloma risk, we conducted a genome-wide association study with validation in additional series totaling 4,692 individuals with multiple myeloma (cases) and 10,990 controls. We identified four risk loci at 3q26.2 (rs10936599, P = 8.70 × 10−14), 6p21.33 (rs2285803, PSORS1C2, P = 9.67 × 10−11), 17p11.2 (rs4273077, TNFRSF13B, P = 7.67 × 10−9) and 22q13.1 (rs877529, CBX7, P = 7.63 × 10−16). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy, as well as insight into the biological basis of predisposition.

Circulating microRNAs in hematological diseases: principles, challenges, and perspectives

The complex microRNA (miRNA) network plays an important role in the regulation of cellular processes such as development, differentiation, and apoptosis. Recently, the presence of cell-free miRNAs that circulate in body fluids was discovered. The ability of these circulating miRNAs to mirror physiological and pathophysiological conditions as well as their high stability in stored patient samples underlines the potential of these molecules to serve as biomarkers for various diseases. In this review, we describe recent findings in miRNA-mediated cell-to-cell communication and the functions of circulating miRNAs in the field of hematology. Furthermore, we discuss current approaches to design biomarker studies with circulating miRNAs. This article critically reviews the novel field of circulating miRNAs and highlights their suitability for clinical and basic research in addition to their potential as a novel class of biomarkers.

Impact of serum storage conditions on microRNA stability

As microRNAs (miRNAs) were found to be present in human serum in a remarkably stable and cell-independent form, their potential as novel noninvasive biomarkers for physiological and pathophysiological conditions, including cancer, is of growing interest.1, 2 Serum miRNAs can be found in exosomes3 and bound to proteins, such as AGO2,4 increasing their stability and circulation time. However, owing to limited space and availability of −80 °C freezers, many hospitals and laboratories store patient serum samples at −20 °C, for many years. We therefore investigated the impact of short-term, intermediate-term and long-term storage at −80 and −20 °C on the stability and abundance of individual and total miRNAs in human serum.