Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies.

BACKGROUND: Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. METHODS: Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. RESULTS: Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. CONCLUSIONS: In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia.

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Droplet Digital PCR Is a Robust Tool for Monitoring Minimal Residual Disease in Adult Philadelphia-Positive Acute Lymphoblastic Leukemia.

The breakpoint cluster region-abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression.

RARA and RARG gene downregulation associated with EZH2 mutation in acute promyelocytic-like morphology leukemia.

Most acute promyelocytic leukemia (APL) patients express PML-RARA fusion; in rare cases, RARA is rearranged with partner genes other than PML. To date, only 2 patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an acute myeloid leukemia patient with morphology resembling APL without involvement of the RARA gene. Molecular and fluorescent in situ hybridization analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci. Targeted next-generation sequencing showed EZH2- D185H mutation. As this mutation involved the region of interaction with DNA methyltransferases, we speculate an epigenetic alteration of genes involved in the APL-like phenotype. Expression analysis by droplet digital polymerase chain reaction revealed downregulation of the RARA and RARG genes. We hypothesize a novel mechanism of EZH2 function alteration, which may be responsible for an acute myeloid leukemia with APL-like phenotype featuring dysregulation of the RARA and RARG genes.

Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology.

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia.

Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse.

Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents.

Hepatic maturation of human fetal hepatocytes in four-compartment three-dimensional perfusion culture.

Bio-artificial liver support systems have been utilized as bridging devices to support acute and chronic liver injury. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and long-term survival of adult cells in vitro. As an alternative cell source, we investigated the potential of human fetal hepatocytes (hFH) in a four-compartment hollow fiber-based three-dimensional (3D) perfusion culture system. hFH were isolated from 17- to 19-gestational-week livers and cultured in the 3D perfusion bioreactors for 14 days. Metabolism activity, hepatocyte-specific gene expression, protein expression, and hepatic function were investigated. Increased glucose consumption and lactate production indicated cell proliferation in the bioreactor. The ratio of cytochrome P450 3A4 to 3A7 gene expression and the increase of the number of asialoglycoprotein receptor-positive cells indicated cell differentiation into mature hepatocytes. Histological and immunohistochemical analysis revealed reorganization of fetal liver cells. Hepatic function was further examined for ammonia metabolism and for albumin production using colorimetric assays and enzyme-linked immunosorbent assay, respectively. In contrast to conventional 2D culture, the 3D perfusion culture system induced functional maturation to hFH; these cells may be useful as an alternative cell source for extracorporeal liver support.

Quantitative comparison of stem cell marker-positive cells in fetal and term human amnion.

Scattered in the amniotic epithelium of the human term placenta are pluripotent stem cell marker-positive cells. Unlike other parts of the placenta, amniotic epithelial (AE) cells are derived from pluripotent epiblasts. It is hypothesized that most epiblast-derived fetal AE cells are positive for stem cell markers at the beginning of pregnancy and that the stem cell marker-positive cells scattered through the term amnion are remaining, epiblast-like stem cells. To test this hypothesis, human fetal amnia from early-stage pregnancies were evaluated for expression of the stem cell-specific cell surface markers TRA 1-60 and TRA 1-81 and of the pluripotent stem cell marker genes Oct4, Nanog, and Sox2. Whole-mount immunohistochemical analysis demonstrated that a greater proportion of AE cells in the 17-19 week human fetal amnion are positive for both TRA 1-60 and TRA 1-81 than in the term amnion. Quantitative real-time RT-PCR analysis confirmed that the fetal AE cells exhibit greater stem cell marker gene expression than those in term placentae. Expression of both Nanog and Sox2 mRNAs were significantly higher in the fetal amnion, while Oct4 mRNA expression was not significantly changed. These differences in abundance of stem cell marker-positive cells and stem cell marker gene expression together indicate that some stem cell marker-positive cells are conserved over the course of pregnancy. The results suggest that stem cell marker-positive AE cells in the term amnion are retained from epiblast-derived fetal AE cells.

Effect of Culture Conditions on Reference Genes Expression in Placenta-derived Stem Cells.

BACKGROUND AND OBJECTIVES: Normalization with valid reference genes is crucial for gene expression analysis with quantitative real-time reverse transcription PCR (qRT-PCR). This is especially relevant when stem cells are investigated with respect to gene expression in the differentiation process. Due to the plasticity of the stem cells, the variation of reference gene expression may cause misinterpretation of the target gene expression. METHODS AND RESULTS: In this study, we investigated the gene expression stability of commonly used 32 reference genes in placenta-derived stem cells, which were cultured with or without exogenous epidermal growth factor. The influence of unstable reference gene expression on the data interpretation was also demonstrated with stem cell marker gene expressions on the placenta-derived stem cells. Statistical validation analysis of reference genes revealed the stability of each gene. Commonly used ß-actin, 18S and GAPDH expression were relatively instable. The cell cycle relating house keeping genes, PPIA, POLR2A, and POP4 were most stable in the compared culture conditions. Reference genes were divided into the following three groups and statistically analyzed; 1) unstable genes, 2) stable genes, and 3) commonly used genes. The results indicate that the interpretation of the experiments was significantly different depending on the stability of the reference genes. CONCLUSIONS: In the stem cell experiments, even minor differences in the culture conditions influenced the expression of reference genes. Thus, the identification of valid reference genes must be determined at each experimental setting. We recommend performing a stepwise screening process to determine valid reference genes.