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The objective of this study was to assess the clinical impact and financial costs of next-generation sequencing (NGS) in 5 categories of pediatric and adult hematological cancers. NGS prescriptions were prospectively collected from 26 laboratories, with varied technical and reporting practice (all or only significant targets). Impact was defined by the identification of (1) an actionable mutation, (2) a mutation with prognostic and/or theranostic value, and/or (3) a mutation allowing nosological refinement, reported by local investigators. A microcosting study was undertaken in 4 laboratories, identifying the types and volumes of resources required for each procedural step. Individual index prescriptions for 3961 patients were available for impact analysis on the management of myeloid disorders (two thirds) and, mainly mature B, lymphoid disorders (one third). NGS results were considered to impact the management for 73.4% of prescriptions: useful for evaluation of prognostic risk in 34.9% and necessary for treatment adaptation (actionable) in 19.6%, but having no immediate individual therapeutic impact in 18.9%. The average overall cost per sample was 191 € for the restricted mature lymphoid amplicon panel. Capture panel costs varied from 369 € to 513 €. Unit costs varied from 0.5 € to 5.7 € per kb sequenced, from 3.6 € to 11.3 € per target gene/hot-spot sequenced and from 4.3 € to 73.8 € per target gene/hot-spot reported. Comparable costs for the Amplicon panels were 5–8 € per kb and 10.5–14.7 € per target gene/hot-spot sequenced and reported, demonstrating comparable costs with greater informativity/flexibility for capture strategies. Sustainable funding of precision medicine requires a transparent discussion of its impact on care pathways and its financial aspects.

Abstract Background We studied a young woman with atypical diabetes associated with mild intellectual disability, lymphedema distichiasis syndrome (LDS) and polymalformative syndrome including distichiasis. We used different genetic tools to identify causative pathogenic mutations and/or copy number variations. Results Although proband’s, diabetes mellitus occurred during childhood, type 1 diabetes was unlikely due to the absence of detectable autoimmunity. DNA microarray analysis first identified a de novo, heterozygous deletion at the chr16q24.2 locus. Previously, thirty-three pathogenic or likely pathogenic deletions encompassing this locus have been reported in patients presenting with intellectual deficiency, obesity and/or lymphedema but not with diabetes. Of note, the deletion encompassed two topological association domains, whose one included FOXC2 that is known to be linked with LDS. Via whole-exome sequencing, we found a heterozygous, likely pathogenic variant in WFS1 (encoding wolframin endoplasmic reticulum [ER] transmembrane glycoprotein) which was inherited from her father who also had diabetes. WFS1 is known to be involved in monogenic diabetes. We also found a likely pathogenic variant in USP9X (encoding ubiquitin specific peptidase 9 X-linked) that is involved in X-linked intellectual disability, which was inherited from her mother who had dyscalculia and dyspraxia. Conclusions Our comprehensive genetic analysis suggested that the peculiar phenotypes of our patient were possibly due to the combination of multiple genetic causes including chr16q24.2 deletion, and two likely pathogenic variants in WFS1 and USP9X.

Molecular tests have become an indispensable tool for the diagnosis and prognosis of hematological malignancies and are subject to accreditation according to the International Standard ISO 15189. National standardization of these techniques is essential to ensure that patients throughout France benefit from the same care. We report here on the experience of the GBMHM (Groupe des Biologistes Moléculaires des Hémopathies Malignes). By organizing External Evaluation of Quality (EEQ) programs and training meetings, the GBMHM has contributed to improvement and standardization of molecular tests in 64 French laboratories. A retrospective analysis of the quality-control results of 11 national campaigns spanning 10 years was performed for the 3 most frequently prescribed tests: BCR-ABL1, JAK2 V617F, and lymphoid clonality. For each test, particular attention was placed on comparing methodologies and their evolution throughout the period. The establishment of the BCR-ABL1, JAK2 V617F, and lymphoid clonality EEQ programs and the associated training meetings have initiated a process of collective standardization concerning the methods of implementation (JAK2 V617F) and the interpretation and formulation of results (lymphoid clonality). In addition, it resulted in objective improvement in technical performance (BCR-ABL1). Our evaluation of the impact of these EEQ programs demonstrates that it is possible to obtain reproducible values across different laboratories in France by applying national recommendations. To our knowledge, this is the first publication that evaluates the impact of a national quality assurance program on improving molecular results in hematology.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P

In t(8;21)(q22;q22) acute myeloid leukemia, the prognostic value of early minimal residual disease assessed with real-time quantitative polymerase chain reaction is the most important prognostic factor, but how long-term minimal residual disease monitoring may contribute to drive individual patient decisions remains poorly investigated. In the multicenter CBF-2006 study, a prospective monitoring of peripheral blood and bone marrow samples was performed every 3 months and every year, respectively, for 2 years following intensive chemotherapy in 94 patients in first complete remission. A complete molecular remission was defined as a (RUNX1-RUNX1T1/ABL1)×100 ≤ 0.001%. After the completion of consolidation therapy, a bone marrow complete molecular remission was observed in 30% of the patients, but was not predictive of subsequent relapse. Indeed, 8 patients (9%) presented a positive bone marrow minimal residual disease for up to 2 years of follow-up while still remaining in complete remission. Conversely, a peripheral blood complete molecular remission was statistically associated with a lower risk of relapse whatever the time-point considered after the completion of consolidation therapy. During the 2-year follow-up, the persistence of peripheral blood complete molecular remission was associated with a lower risk of relapse (4-year cumulative incidence, 8.2%), while molecular relapse confirmed on a subsequent peripheral blood sample predicted hematological relapse (4-year cumulative incidence, 86.9%) within a median time interval of 3.9 months. In t(8;21)(q22;q22) acute myeloid leukemia, minimal residual disease monitoring on peripheral blood every 3 months allows for the prediction of hematological relapse, and to identify patients who could potentially benefit from intervention therapy.

Core-binding factor acute myeloid leukemia is a favorable acute myeloid leukemia subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, disrupting RUNX1 (previously CBFA/AML1) or CBFB transcription factor functions. The receptor tyrosine kinase KIT is expressed in the vast majority of these acute myeloid leukemias and frequent activating KIT gene mutations have been associated with a higher risk of relapse. This phase II study aimed to evaluate dasatinib as maintenance therapy in patients with core-binding factor acute myeloid leukemia in first hematologic complete remission, but at higher risk of relapse due to molecular disease persistence or recurrence. A total of 26 patients aged 18–60 years old previously included in the CBF-2006 trial were eligible to receive dasatinib 140 mg daily if they had a poor initial molecular response (n=18) or a molecular recurrence (n=8). The tolerance of dasatinib as maintenance therapy was satisfactory. The 2-year disease-free survival in this high-risk population of patients was 25.7%. All but one patient with molecular recurrence presented subsequent hematologic relapse. Patients with slow initial molecular response had a similar disease-free survival when treated with dasatinib (40.2% at 2 years) or without any maintenance (50.0% at 2 years). The disappearance of KIT gene mutations at relapse suggests that clonal devolution may in part explain the absence of efficacy observed with single-agent dasatinib in these patients (n. EudraCT: 2006-006555-12).

In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex‐FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug‐resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)). Colour figure can be viewed on http://www.esacp.org/acp/2003/25‐3/struski.htm.

Early response to chemotherapy has a major prognostic impact in acute myeloid leukemia patients treated with a double induction strategy. Less is known about patients treated with standard-dose cytarabine and anthracycline. We designed a risk-adapted remission induction regimen in which a second course of intermediate-dose cytarabine was delivered after standard “7+3” only if patients had 5% or more bone marrow blasts 15 days after chemotherapy initiation (d15-blasts). Of 823 included patients, 795 (96.6%) were evaluable. Five hundred and forty-five patients (68.6%) had less than 5% d15-blasts. Predictive factors for high d15-blasts were white blood cell count (P

It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor EVI1 is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 de novo AML patients for the expression of DNMT3A, 3B (and its non-catalytic variant 3B(NC)) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of DNMT3B/3B(NC) is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and DNMT3A expression level. Moreover, multivariate analysis showed that a high expression level of DNMT3B/3B(NC) is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of DNMT3B/3B(NC) is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.

Background Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34+CD38low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34+CD38low/−CD123+ phenotype.Design and Methods The percentage of CD34+CD38low/−CD123+ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses.Results A proportion of CD34+CD38low/−CD123+ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34+CD38low/−CD123+ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34+CD38low/−CD123+ cells negatively affected disease-free survival (0.9 versus 4.7 years; P

Ten-eleven translocation 2 (TET2) mutations have been involved in myeloid malignancies. This retrospective study aims at evaluating the frequency and impact of TET2 mutations in 247 secondary acute myeloid leukemia cases referred to as myelodysplasia-related changes (n=201) or therapy-related (n=46) leukemias. Mutation of at least one copy of the TET2 gene was detected in 49 of 247 (19.8%) patients who presented with older age, higher hemoglobin level, higher neutrophil and monocyte counts, and lower platelet count. TET2 mutations were significantly less frequent in therapy-related (8.7%) than myelodysplasia-related changes (22.3%; P=0.035) leukemias and strongly associated with normal karyotype (P

The PI3K/AKT and mTOR signaling pathways are activated in acute myeloid leukemia, including in the more immature leukemic populations. Constitutive PI3K activation is detectable in 50% of acute myeloid leukemia samples whereas mTORC1 is activated in all cases of this disease. In leukemic cells, the PI3K activity relates to the expression of the p110δ isoform of class IA PI3K. Constitutive PI3K activation is the result of autocrine IGF-1/IGF-1R signaling in 70% of acute myeloid leukemia samples but specific inhibition of this pathway does not induce apoptosis. Specific inhibition of PI3K/AKT or mTORC1 alone in vitro has anti-leukemic effects which are essentially exerted via the suppression of proliferation. However, as mTORC1 activation is independent of PI3K/AKT in acute myeloid leukemia, dual PI3K and mTOR inhibitors may induce apoptosis in blast cells. Moreover, mTORC1 inhibition using sirolimus overactivates PI3K/AKT via the upregulation of IRS2 expression and by favoring IGF-1/IGF-1R autocrine signaling. Recent data also indicate that mTORC1 does not control protein translation in acute myeloid leukemia. These results open the way for the design of direct inhibitors of protein synthesis as novel acute myeloid leukemia therapies and also for the development of second generation mTOR inhibitors (the TORKinhibs).

Background Alterations in the PI3K/Akt pathway are found in a wide range of cancers and the development of PI3K inhibitors represents a promising approach to cancer therapy. Constitutive PI3K activation, reflecting an intrinsic oncogenic deregulation of primary blast cells, is detected in 50% of patients with acute myeloid leukemia. However, the mechanisms leading to this activation are currently unknown. As we previously reported IGF-1 autocriny in acute myeloid leukemia cells, we investigated whether IGF-1 signaling was involved in the constitutive activation of PI3K.Design and Methods We analyzed the IGF-1/IGF-1R signaling pathway and PI3K activity in 40 acute myeloid leukemia bone marrow samples. Specific inhibition of IGF-1/IGF-1R signaling was investigated using neutralizing anti-IGF-1R, anti-IGF-1 antibodies or IGF-1 short interfering RNA. The anti-leukemic activity of the neutralizing anti-IGF-1R was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation and survival.Results In all samples tested, we found that functional IGF-1R was constantly expressed in leukemic cells. In the acute myeloid leukemia samples with PI3K activation, we found that the IGF-1R was constitutively phosphorylated, although no IGF-1R activating mutation was detected. Specific inhibition of IGF-1R signaling with neutralizing anti-IGF-1R strongly inhibited the constitutive phosphorylation of both IGF-1R and Akt in 70% of the PI3K activated samples. Moreover, both incubation with anti-IGF-1 antibody and IGF-1 short interfering RNA inhibited Akt phosphorylation in leukemic cells. Finally, neutralizing anti-IGF-1R treatment decreased the clonogenicity of leukemic progenitors and the proliferation of PI3K activated acute myeloid leukemia cells.Conclusions Our current data indicate a critical role for IGF-1 autocriny in constitutive PI3K/Akt activation in primary acute myeloid leukemia cells and provide a strong rationale for targeting IGF-1R as a potential new therapy for this disease.

Abstract Background Chronic lymphocytic leukemia (CLL) cells are often affected by genomic aberrations targeting key regulatory genes. Although fludarabine is the standard first line therapy to treat CLL, only few data are available about the resistance of B cells to this purine nucleoside analog in vivo. Here we sought to increase our understanding of fludarabine action and describe the mechanisms leading to resistance in vivo. We performed an analysis of genomic aberrations, gene expression profiles, and microRNAs expression in CLL blood B lymphocytes isolated during the course of patients' treatment with fludarabine. Results In sensitive patients, the differentially expressed genes we identified were mainly involved in p53 signaling, DNA damage response, cell cycle and cell death. In resistant patients, uncommon genomic abnormalities were observed and the resistance toward fludarabine could be characterized based on the expression profiles of genes implicated in lymphocyte proliferation, DNA repair, and cell growth and survival. Of particular interest in some patients was the amplification of MYC (8q) observed both at the gene and transcript levels, together with alterations of myc-transcriptional targets, including genes and miRNAs involved in the regulation of cell cycle and proliferation. Differential expression of the sulfatase SULF2 and of miR-29a, -181a, and -221 was also observed between resistant and sensitive patients before treatment. These observations were further confirmed on a validation cohort of CLL patients treated with fludarabine in vitro. Conclusion In the present study we identified genes and miRNAs that may predict clinical resistance of CLL to fludarabine, and describe an interesting oncogenic mechanism in CLL patients resistant to fludarabine by which the complete MYC-specific regulatory network was altered (DNA and RNA levels, and transcriptional targets). These results should prove useful for understanding and overcoming refractoriness to fludarabine and also for predicting the clinical outcome of CLL patients before or early during their treatment.

Defects in the fusion of facial buds can result from an anomaly in tissue development or apoptosis, or both. Our working hypothesis was that anomalies in the development of tissues could be caused by a genetic angiogenic defect. Our main objective was to design a reproducible experimental model to study the expression of angiogenic genes in the borders of cleft lips with or without cleft palate. We therefore prospectively studied seven non-syndromic patients, three with a cleft lip (2 right, 1 left), and four with a cleft lip and palate (1 bilateral, 2 right, 1 left), with no CGH (comparative genomic hybridisation) array, who had primary operations to repair their clefts. We also used four controls (cultured fibroblasts from healthy skin samples). The mean (range) age at operation was 44 (13-77) days. We studied the lateral and medial borders histologically and did qPCR (quantitative real-time polymerase chain reaction) analysis for gene expression with 22 genes of interest (and two housekeeping genes) involved in cleft lip and angiogenesis. The qPCR analysis found significant (p < 0.05) overexpression of eight genes in the medial border and seven in the lateral border, and underexpression of nine genes in the medial, and ten in the lateral border. The difference in expression between the two borders was not significant. This preliminary study has enabled us to develop a new method to analyse the expression of angiogenic genes in the borders of cleft lips. [ABSTRACT FROM AUTHOR]

Summary: Rituximab, an anti-CD20 monoclonal antibody, is widely used in the treatment of B-cell lymphoma. Some reports have outlined histologic modifications in bone marrow specimens from patients treated with this antibody, notably the presence of CD3+ lymphoid aggregates morphologically mimicking residual lymphoma. To gain insight into the significance of such infiltrates, serial BM trephines obtained in 39 patients with B-cell follicular lymphoma treated by rituximab and enrolled in the GOELAMS-GELA intergroup FL2000 protocol were reexamined. The 39 patients were 22 women and 17 men with a median age of 50 years (range, 29-75 years). All pretreatment bone marrow biopsies showed CD20+ lymphomatous cells. A second biopsy was obtained between 30 and 100 days after the last rituximab injection: 19 (48%) were morphologically diagnosed as negative (no lymphoid infiltrates or only minor lymphoid aggregates) and 20 (51%) as positive because of persistent lymphoid nodules. After immunohistochemical analysis, 13 (33%) cases were reinterpreted as false-positive because of the complete absence of CD20+ cells, with the lymphoid nodules consisting of CD3+ and CD5+ T cells. Most of them also expressed CD4+, whereas only a few CD8+ cells were present. Among these 13 false-positive cases, 12 were BCL2-IGH polymerase chain reaction–negative in the bone marrow aspirate at the time of biopsy. The 13th case turned out to be negative in the 18th-month bone marrow aspirate. In all of these cases, lymphoid aggregates had disappeared on bone marrow biopsies performed 18 months after treatment. After a mean follow-up of 4.5 years, 9 of 13 patients were in remission as compared with only 2 among the 7 patients with postrituximab persistent CD20+ lymphomatous cells. There was no statistically significant difference between this false-positive group of patients and that with negative postrituximab bone marrow regarding sex, age, medullar involvement pattern before treatment, delay between rituximab treatment, and molecular status. Interestingly, we noted a more favorable outcome (70% versus 52% remission) for the false-positive cases, suggesting that these T-cell reactions could be the hallmark of specific antitumoral immunity after rituximab treatment and should be properly investigated. [Copyright &y& Elsevier]