One-Step Duplex Droplet Digital PCR for WT1 Overexpression.

With the improvement of treatment methods in acute hematology malignancies, the development of sensitive tools for minimal residual disease assessment has become a priority. The monitoring of WT1 expression level by real-time quantitative PCR has been a standard for minimal residual disease evaluation in acute myeloid leukemia and, since 2009, has been optimized through a European LeukemiaNet effort in an established protocol with well-defined clinical end points. Building on the work of the European LeukemiaNet, this article reports the development of a novel, one-step duplex WT1/ABL1 droplet digital assay for WT1 overexpression detection. This assay provides accurate data with high precision and linearity, even at low-template concentration, while retaining strong correlation with the standardized method and therefore maintaining the framework to analyze the results in the context of acute myeloid leukemia patients.

Undetectable circulating tumor DNA (ctDNA) levels correlate with favorable outcome in metastatic melanoma patients treated with anti-PD1 therapy.

BACKGROUND: Treatment with anti-PD1 monoclonal antibodies improves the survival of metastatic melanoma patients but only a subgroup of patients benefits from durable disease control. Predictive biomarkers for durable benefit could improve the clinical management of patients. METHODS: Plasma samples were collected from patients receiving anti-PD1 therapy for ctDNA quantitative assessment of BRAFV600 and NRASQ61/G12/G13 mutations. RESULTS: After a median follow-up of 84 weeks 457 samples from 85 patients were analyzed. Patients with undetectable ctDNA at baseline had a better PFS (Hazard ratio (HR) = 0.47, median 26 weeks versus 9 weeks, p = 0.01) and OS (HR = 0.37, median not reached versus 21.3 weeks, p = 0.005) than patients with detectable ctDNA. Additionally, the HR for death was lower after the ctDNA level became undetectable during follow-up (adjusted HR: 0.16 (95% CI 0.07-0.36), p-value < 0.001). ctDNA levels > 500 copies/ml at baseline or week 3 were associated with poor clinical outcome. Patients progressive exclusively in the central nervous system (CNS) had undetectable ctDNA at baseline and at subsequent assessments. In multivariate analysis adjusted for LDH, CRP, ECOG and number of metastatic sites, the ctDNA remained significant for PFS and OS. A positive correlation was observed between ctDNA levels and total metabolic tumor volume (TMTV), number of metastatic sites and total tumor burden. CONCLUSIONS: Assessment of ctDNA baseline and during therapy was predictive for tumor response and clinical outcome in metastatic melanoma patients and reflected the tumor burden. ctDNA evaluation provided reliable complementary information during anti-PD1 antibody therapy.

Circulating tumor DNA in early response assessment and monitoring of advanced colorectal cancer treated with a multi-kinase inhibitor.

Predictive biomarkers are eagerly awaited in advanced colorectal cancer (aCRC). Targeted sequencing performed on tumor and baseline plasma samples in 20 patients with aCRC treated with regorafenib identified 89 tumor-specific mutations of which ≥50% are also present in baseline plasma. Droplet digital PCR (ddPCR) assays were optimized to monitor circulating tumor DNA (ctDNA) levels in plasmatic samples collected throughout the treatment course and showed the importance of using the absolute value for ctDNA rather than the mutant/wild type ratio in monitoring the therapy outcome. High baseline cell free DNA (cfDNA) levels are associated with shorter overall survival (OS) (HR 7.38, P=0.001). An early increase (D14) in mutated copies/mL is associated with a significantly worse PFS (HR 6.12, P=0.008) and OS (HR 8.02, P=0.004). These data suggest a high prognostic value for early ctDNA level changes and support the use of blood-born genomic markers as a tool for treatment.

Identification of chronic myeloid leukemia patients treated with imatinib who are potentially eligible for treatment discontinuation by assessing real-life molecular responses on the international scale in a EUTOS-certified lab.

A retrospective study was performed to describe molecular responses (MR) on the international scale (IS) in patients with chronic myeloid leukemia (CML) treated with imatinib in routine clinical practice in Belgium and to identify patients potentially eligible for treatment discontinuation. The analysis included 116 patients with CML in chronic phase at treatment centers sending blood samples for molecular follow-up to a single EUTOS-certified laboratory. IS MR from the last patient visit between October 2014 and April 2015 were retrospectively collected. Most patients (93.1%) had an IS MR corresponding to an optimal response per European LeukemiaNet 2013 guidelines; 53.4% (62/116) of patients were in deep molecular responses ≥MR4.5 at their last visit (mean treatment duration: 91.0 months) among whom 36.2% (42/116) had been receiving imatinib for >5.8 years and 26.7% (31/116) for >8 years (margins of error: 8.74% and 8.05%, respectively). These patients would likely have the highest chance of staying in treatment-free remission (TFR) upon discontinuation, based on published TFR trial data. Although our study only provides a snapshot in time of a patient's last MR reported, without precise information regarding MR duration, the study settings could nevertheless support the feasibility of attempting TFR outside clinical trials in the future.

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.

Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples.

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy.

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.

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.

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.

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.

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.

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.

Farnesyl transferase inhibitor (lonafarnib) in patients with myelodysplastic syndrome or secondary acute myeloid leukaemia: a phase II study.

Although an activating mutation of Ras is commonly observed in myelodysplastic syndrome (MDS), the role of Ras in the natural history of MDS remains largely unknown. We prospectively studied efficiency and tolerance of lonafarnib, a compound able to inhibit Ras signalling pathway through an inhibition of farnesyl transferase, in patients with MDS or secondary acute myeloid leukaemia (sAML). Lonafarnib was administered orally at a dose of 200 mg twice daily for three courses of 4 weeks (separated by 1 to 4 weeks without treatment). Sixteen patients were included: FAB/RAEB (n = 10), RAEB-T (n = 2), sAML (n = 2) and chronic myelomonocytic leukaemia (CMML; n = 2); WHO/RAEB-1 (n = 4), RAEB-2 (n = 5), AML (n = 5), CMML (n = 2). Median age was 70 (53-77) years. The karyotype was complex or intermediate in 11 patients, and the International Prognostic Scoring Systems (IPSS) risk groups were low in two patients, INT-1 in one patient, INT-2 in four patients and high in six patients (unknown or not applicable in three patients). Among the 14 patients tested, five had Ras mutations in codons 12, 13 or 61 of N-Ras, K-Ras or H-Ras. One patient was excluded of the analysis for protocol violation, and 15 patients were assessable for tolerance. Gastrointestinal toxicities (diarrhoea, nausea and anorexia) and myelosuppression were the major side effects. Other toxicities included infections, fatigue, increase of liver enzymes, arrhythmia and skin rash. One patient died of infection, and the treatment was stopped in one other who developed atrial fibrillation. Doses were reduced in all but one patient treated with more than one course of farnesyl transferase inhibitor. Responses were assessable in 12 patients. A partial response in one sAML patient and a very transient decrease of blast cell count with normalisation of karyotype in one MDS patient were observed. No relation between improvement of marrow parameters and detected Ras mutations was observed. Lonafarnib alone, administered following our schedule, has shown limited activity in patients with MDS or secondary AML. Gastrointestinal and haematological toxicities appear the limiting toxicity in this population of patients.

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) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS 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 ATPgammaS 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 ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, 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 ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, 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 ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.

Chromosomal translocations as a mechanism of BRAF activation in two cases of large congenital melanocytic nevi.

Genetic studies of melanocytic tumors have mainly demonstrated activation of oncogenes such as NRAS or BRAF through point mutations. In two cases of large congenital melanocytic nevi, we observed a chromosomal translocation involving the BRAF oncogene on chromosome 7q34, resulting in both cases in removal of the auto-inhibitory N-terminal regulatory domain (hence the Ras-guanosine triphosphate binding domain) of BRAF from its protein kinase domain. This is early evidence of BRAF activation through chromosomal translocation in melanocytic tumors. Because BRAF point mutations are rather rare in congenital melanocytic nevi and melanoma arising in non-sun-exposed area, the molecular mechanism of oncogenic activation as described here could be a recurrent molecular feature in these groups of melanocytic tumors.

Limitations and practical procedure in BclII-Ig heavy chain gene rearrangement real-time quantitative polymerase chain reaction.

Follicular lymphoma is characterized by the t(14;18)(q32;q21) translocation, which juxtaposes Ig heavy chain gene (IgH) sequences with the BclII gene. Several publications have highlighted the importance of molecular follow-up in follicular lymphoma, demonstrating that the detection of cells bearing the BclII-IgH rearrangement by real-time quantitative polymerase chain reaction (RQ-PCR) can anticipate a clinical relapse. In this context, we developed a BclII-IgH RQ-PCR. We began with SYBR Green I detection technology but observed that this system does not allow an accurate measurement of the tumor load when working with genomic DNA. While we were designing the assay using Taqman technology, Moppett et al (Moppett J, van der Velden VHJ, Wijkhuijs AJM, Hancock J, van Dongen JJM, Goulden N: Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 2003, 17:268-270) reported PCR inhibition problems in around 15% of blood and bone marrow samples, affecting the DNA quantification and thus the assessment of minimal residual disease. They demonstrated that this PCR inhibition could be partially resolved by adding nonacetylated bovine serum albumin. In our studies, we observed the same phenomenon in a single follicular lymphoma case and extended our study to other available cases. As a result, we suggest a new RQ-PCR procedure that is based on Taqman probe technology and that takes into account the PCR inhibition problems, making this assay more reliable in a routine molecular laboratory.

Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve.

Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.