Frédéric Leprêtre

Deletion 18q21.2q21.32 involving TCF4 in a boy diagnosed by CGH-array.

We report on a 12 year-old boy presenting with severe developmental delay, dysmorphic features, limb anomalies, growth retardation, hypoplastic vermis and corpus callosum. Conventional and high-resolution cytogenetic analyses were normal. CGH-array allowed characterisation of a de novo 6.2 Mb 18q21.2q21.32 interstitial deletion involving TCF4, the recently identified gene responsible for Pitt-Hopkins syndrome (PHS). No tachypnoea-apnoea paroxysms were observed. We discuss the dysmorphic features particularly involving the ears, which might be helpful towards PHS and 18q21 deletion diagnosis.

Involvement of the immune system, endocytosis and EIF2 signaling in both genetically determined and sporadic forms of Parkinson's disease

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is a common genetic cause of Parkinsons disease (PD). Although patients with sporadic PD and individuals with LRRK2-linked PD display the classical PD phenotype, it is not known whether or not the same biological pathways are deregulated in each context. By using transcriptome profiling, we investigated the deregulation of various biological pathways in a total of 47 peripheral blood mononuclear cell (PBMC) samples from patients with sporadic PD, patients heterozygous for the LRRK2 G2019S mutation compared to healthy controls. We found that the deregulation patterns were indeed similar in PBMCs obtained from patients with sporadic PD and from LRRK2 G2019S carriers, with dysfunctions in mitochondrial pathways, cell survival signaling, cancerization, endocytosis signaling and iron metabolism. Analysis of our PBMC data and other publicly available transcriptome datasets (for whole blood samples) showed that deregulation of the immune system, endocytosis and eukaryotic initiation factor 2 (EIF2) signaling are the main features of transcriptome profiles in PD (since they are also present in the transcriptome of dopaminergic neurons from patients). Transcriptome analysis of PBMCs is thus valuable for (i) characterizing the pathophysiological pathways shared by genetic and sporadic forms of PD and (ii) identifying potential biomarkers and therapeutic targets. This minimally invasive approach opens up tremendous perspectives for better diagnosis and therapy of neurodegenerative diseases because it can be applied from the earliest stages of the disease onwards.

Neurofibromatosis-1 gene deletions and mutations in de novo adult acute myeloid leukemia.

Germline heterozygous alterations of the tumor‐suppressor gene neurofibromatosis‐1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real‐time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1‐deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1‐deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression. Am. J. Hematol. 88:306–311, 2013.

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor‐like state

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL‐60, U266) derived from hematopoietic malignancies contain not only CD34− but also CD34+ subpopulations. The CD34+ cells displayed a stem/progenitor‐like phenotype since, in contrast to CD34− cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose‐based medium. Strikingly, a constant fraction of the CD34+ and CD34− cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz‐scid/scid mice. Cultures in vitro showed that the proportion of CD34+ stem/progenitor‐like cells in the population was decreased by cell–cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34+ cells and overall colony‐forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34+ cells can provide an in vitro model for studies of cancer stem/progenitor cells. J. Cell. Biochem. 112: 1277–1285, 2011.

Characterization by array-CGH of an interstitial de novo tandem 6p21.2p22.1 duplication in a boy with epilepsy and developmental delay.

We report on a 6-years-old boy with psychomotor retardation, mild dysmorphic features and behavioral disturbances associated with epilepsy. Conventional cytogenetic analysis concluded to an interstitial de novo 6p21.2p22.3 duplication. Molecular cytogenetic analysis, including array-CGH technology, allows characterization of this 7.3Mb interstitial tandem duplication. The phenotype of this small 6p duplication reported to date is compared to other cases in the literature. Presence of epilepsy, although rare in patients with 6p duplication may be linked to genes involved in brain function and synaptic transmission in the 6p21.2p22.1 duplicated region (GABBR1, BRD2 and GRM4).

BAP1 Is Altered by Copy Number Loss, Mutation, and/or Loss of Protein Expression in More Than 70% of Malignant Peritoneal Mesotheliomas

Introduction Malignant mesothelioma is a deadly disease that is strongly associated with asbestos exposure. Peritoneal mesotheliomas account for 10% of all the cases. BRCA1 associated protein 1 (BAP1) is a deubiquitinating hydrolase that plays a key role in various cellular processes. Germline and somatic inactivation of BRCA1 associated protein 1 gene (BAP1) is frequent in pleural mesothelioma; however, little is known about its status in peritoneal mesothelioma. Methods Taking advantage of the extensive French National Network for the Diagnosis of Malignant Pleural Mesothelioma and Rare Peritoneal Tumors and the French National Network for the Treatment of Rare Peritoneal Surface Malignancies, we collected biological material and clinical and epidemiological data for 46 patients with peritoneal mesothelioma. The status of BAP1 was evaluated at the mutational and protein expression levels and combined with our previous data on copy number alterations assessed in the same samples. Results We detected mutations in 32% of the malignant peritoneal mesotheliomas analyzed. In addition, we have previously reported that copy number losses occurred in 42% of the samples included in this series. Overall, 73% of the malignant peritoneal mesotheliomas analyzed carried at least one inactivated BAP1 allele, but only 57% had a complete loss of its protein nuclear expression. Better overall survival was observed for patients with BAP1 mutations (p = 0.04), protein expression loss (p = 0.016), or at least one of these alterations (p = 0.007) independently of tumor histological subtype, age, and sex. Conclusions As in pleural mesothelioma, inactivation of BAP1 is frequent in peritoneal mesotheliomas. We found that BAP1 protein nuclear expression is a good prognostic factor and a more reliable marker for the complete loss of BAP1 activity than mutation or copy number loss.

The stem cell-associated gene expression signature allows risk stratification in pediatric acute myeloid leukemia.

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64–6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38–8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63–7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16–7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.

Optimization of routine testing for MET exon 14 splice site mutations in non-small cell lung cancer patients

Introduction: Genomic alterations affecting splice sites of MNNG HOS transforming gene (MET) exon 14 were recently identified in NSCLC patients. Objective responses to MET tyrosine kinase inhibitors have been reported in these patients. Thus, detection of MET exon 14 splice site mutations represents a major challenge. So far, most of these alterations were found by full‐exome sequencing or large capture‐based next‐generation sequencing (NGS) panels, which are not suitable for routine diagnosis. Methods: Aiming to provide a molecular testing method applicable in routine practice, we first developed a fragment‐length analysis for detecting deletions in introns flanking MET exon 14. Second, we designed an optimized targeted NGS panel called CLAPv1, covering the MET exon 14 and flanking regions in addition to the main molecular targets usually covered in genomic testing. In patients with MET exon 14 mutations, MET gene amplification, gene copy number and MET receptor expression were also determined. Results: Among 1514 formalin‐fixed paraffin‐embedded NSCLC samples, nonoptimized NGS allowed detection of MET exon 14 mutations in only 0.3% of the patients, and fragment length analysis detected deletions in 1.1% of the patients. Combined, the optimized CLAPv1 panel and fragment‐length analysis implemented for routine molecular testing revealed MET exon 14 alterations in 2.2% of 365 additional NSCLC patients. MET gene amplification or high gene copy number was observed in 6 of 30 patients (20%) harboring MET exon 14 mutations. Conclusions: These results show that optimized targeted NGS and fragment‐length analysis improve detection of MET alterations in routine practice.

Abstract 3216: Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line

In acute myeloid leukemia (AML), clonal heterogeneity at diagnosis and the presence of minor sub-clones were shown to be responsible for minimal residual disease and relapses in patients. Several studies have proposed that their persistence could be due to their dormant state (i.e. quiescence and/or control of their growth by immune cells). As contribution of immunity to clonal persistence could not be evaluated in xenografts models, we assessed for the clonal diversity of a murine AML cell line. Genomic analyses revealed heterogeneity among the parental cell line and its derived clones. Injected individually into immune-deficient mice, all clones induced AML with different kinetics. However, when administered into immune-competent animals, some clones triggered AML with no mice survival, whereas others elicited reduced lethality rates. The leukemia-surviving mice presented an efficient anti-tumoral CTL activity and reduced lymphopenia. Investigating for residual leukemic cells in the organs of these mice, elimination rather than persistence was observed. Thus, we showed that genetic heterogeneity among the clones led to differential immunogenicity resulting either in their immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients. Citation Format: Carine Brinster, Virginie DRISS, Bruno Quesnel, Martin Figeac, Frederic Lepretre, Celine Villenet, Isabelle Briche. Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2015-3216