Hakim El Housni

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Genotypic and Gene Expression Studies in Congenital Melanocytic Nevi: Insight into Initial Steps of Melanotumorigenesis

Large congenital melanocytic nevi (CMNs) are said to have a higher propensity to malignant transformation compared with acquired nevi. Thus, they represent a good model for studying initial steps of melanotumorigenesis. We have performed genotypic (karyotype, fluorescence in situ hybridization, and mutational analyses) and differential expression studies on a large cohort of medium (n=3) and large (n=24) CMN. Chromosomal abnormalities were rare and single, a feature probably reflecting the benignity of these lesions. Mutational screening showed a high frequency of NRAS mutations in our series (19/27 cases, 70%), whereas BRAF mutations were less common (4/27 cases, 15%). Differential did not show significant alterations of cellular processes such as cell proliferation, cell migration/invasion, angiogenesis, apoptosis, and immune/inflammatory responses. However, significant downregulation of genes involved in pigmentation and upregulation of genes playing a role in DNA protection were observed. Lastly, our microarrays displayed upregulation of genes mediating chemoresistance in cancer. As alteration of pigmentation mechanisms can trigger oxidative damage, increased expression of genes involved in maintenance of DNA integrity might reflect the ability of nevocytic cells to self-protect against cellular stress. Furthermore, the observed alterations linked to chemoresistance might partially account for the well-known inefficacy of chemotherapy in malignant melanoma.

TCF7L2 Polymorphism Associates with New-Onset Diabetes after Transplantation

New-onset diabetes after transplantation (NODAT) is a serious and frequent complication in transplant recipients. Whether NODAT shares the same susceptibility genes as type 2 diabetes is unknown. In this multicenter study, we genotyped 1076 white patients without diabetes at transplantation for 11 polymorphisms that associate with type 2 diabetes. We defined NODAT as a fasting plasma glucose > or =126 mg/dl on at least two occasions or de novo hypoglycemic therapy. We compared clinical and genetic factors between patients who developed NODAT within 6 mo of transplantation (n = 118; incidence 11%) and patients without diabetes (n = 958). In multivariate analysis, NODAT significantly associated with the following characteristics: TCF7L2 polymorphism (odds ratio [OR] 1.60 per each T allele; P = 0.002), age (OR 1.03 per year; P < 0.001), body mass index at transplantation (OR 1.09 per unit; P < 0.001), tacrolimus use (OR 2.26; P < 0.001), and the occurrence of a corticoid-treated acute rejection episode (OR 2.78; P < 0.001). In summary, our data show that the TCF7L2 rs7903146 polymorphism, a known risk factor for type 2 diabetes in the general population, also associates with NODAT.

Clinical Validation of Targeted Next Generation Sequencing for Colon and Lung Cancers

Objective Recently, Next Generation Sequencing (NGS) has begun to supplant other technologies for gene mutation testing that is now required for targeted therapies. However, transfer of NGS technology to clinical daily practice requires validation. Methods We validated the Ion Torrent AmpliSeq Colon and Lung cancer panel interrogating 1850 hotspots in 22 genes using the Ion Torrent Personal Genome Machine. First, we used commercial reference standards that carry mutations at defined allelic frequency (AF). Then, 51 colorectal adenocarcinomas (CRC) and 39 non small cell lung carcinomas (NSCLC) were retrospectively analyzed. Results Sensitivity and accuracy for detecting variants at an AF >4% was 100% for commercial reference standards. Among the 90 cases, 89 (98.9%) were successfully sequenced. Among the 86 samples for which NGS and the reference test were both informative, 83 showed concordant results between NGS and the reference test; i.e. KRAS and BRAF for CRC and EGFR for NSCLC, with the 3 discordant cases each characterized by an AF <10%. Conclusions Overall, the AmpliSeq colon/lung cancer panel was specific and sensitive for mutation analysis of gene panels and can be incorporated into clinical daily practice.

A Real-Time Polymerase Chain Reaction Assay for Rapid, Sensitive, and Specific Quantification of the JAK2V617F Mutation Using a Locked Nucleic Acid-Modified Oligonucleotide

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

Single-Nucleotide Polymorphism Genotyping by Melting Analysis of Dual-Labeled Probes: Examples Using Factor V Leiden and Prothrombin 20210A Mutations

The emergence of fluorescence techniques in 96- or 384-well formats has led to high-throughput single-nucleotide polymorphism (SNP) detection methods. Among these, allele discrimination based on real-time PCR technologies has been developed along two main methodologic approaches. In the first approach, a temperature-dependent fluorescent signal is generated by hybridization of a probe at the end of each PCR cycle. The monitoring of fluorescence emission is followed during (1)(2) or at the end of (3)(4) the PCR process. In the latter case, allele detection is achieved by carrying out of a melting curve analysis. In the second approach, exploiting the classic 5′ nuclease assay (TaqMan®) technology, a temperature-independent fluorescence signal is generated during the PCR process by the exonuclease degradation of a hybridized TaqMan probe (5)(6). Except for one method discussed later (3), all of these techniques require the use of two probes for allele differentiation. An allele differentiation study has stressed the risk of artifacts and subsequent erroneous results when a polymorphism is located within the DNA sequence targeted by TaqMan (7). The same study demonstrated the power of methods relying on determination of melting curves. Although other interesting approaches have been reported recently, such as DASH (8), iFret (9), and dipole calculation (10), some of them are technically difficult or time-consuming. In addition, the SYBR Green technique (11) would represent the simplest method to detect point mutations, but it seems to be limited by its lack of ability to differentiate alleles (12). We present here a new methodologic approach that allows high-throughput genotyping of SNPs. Allele differentiation is achieved by the generation of melting curves, with a single dual-labeled probe, at the end of a classic PCR reaction. The reliability of the method is demonstrated for detection of the factor V Leiden …

Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPγS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPγS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPγS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPγS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPγS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPγS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPγS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPγS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.

Cloning and sequence analysis of human calcyphosine complementary DNA

Calcyphosine, initially identified as thyroid protein p24, is a calcium-binding protein containing four EF-hand domains. It was first cloned and characterized in the dog and corresponds to R2D5 antigen in rabbit. Using the canine calcyphosine cDNA sequence as a probe, we have isolated its human counterpart from a thyroid cDNA library. The two sequences display a high degree of conservation, both at nucleotide and deduced amino acid levels. Sequence comparison with other proteins showed that the closest homologue of calcyphosine is the crustacean CCBP-23 protein. Northern blot analysis revealed that calcyphosine messenger RNA is much less abundant in human than in canine thyrocytes. Western blot experiments indicated that the amount of protein is also dramatically reduced in man compared to dog.

Global histone deacetylase enzymatic activity is an independent prognostic marker associated with a shorter overall survival in chronic lymphocytic leukemia patients

Histone deacetylases (HDAC) play a crucial role in transcriptional regulation and are often deregulated in many cancers. However, global HDAC enzymatic activity has never been investigated in Chronic Lymphocytic Leukemia (CLL). We measured HDAC activity in protein extracts from CD19+ B-cells purified from 114 CLL patients with a median follow-up of 91 months (range: 11–376). HDAC activity was equivalent in CLL and normal B-cells but higher in patients who died during the study than in living patients (152.1 vs. 65.04 pmol; P = 0.0060). Furthermore, HDAC activity correlated with treatment-free survival (TFS; P = 0.0156) and overall survival (OS; P < 0.0001): patients with low HDAC activity (n = 75) had a median TFS and OS of 101 and >376 months, respectively, whereas patients with high HDAC activity (n = 39) had a median TFS and OS of 47 and 137 months, respectively. Multivariate analyses indicated that HDAC activity is an independent predictor of OS (hazard ratio = 7.68; P = 0.0017). Finally, HDAC activity increased after B-cell receptor stimulation using IgM, suggesting a role for microenvironment stimuli (n = 10; P = 0.0371). In conclusion, high HDAC activity in CLL B-cells is associated with shorter TFS and OS and is an independent marker of OS, refining the use of other prognostic factors. This work provides a biological base for the use of HDAC inhibitors in CLL treatment.

Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations’ monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.