Analysis of N-nitrosodimethylamine in metformin hydrochloride products by high-resolution accurate mass gas chromatography mass spectrometry.

RATIONALE: The high resolving power of the Orbitrap mass spectrometer in a high-resolution accurate mass gas chromatography (HRAM-GC-MS) system provides greater selectivity and sensitivity for the identification and quantification of volatile analytes at low parts per billion (ppb) levels. Hence, it can be applied for the analysis of pharmaceutical impurities like N-nitrosodimethylamine (NDMA) in metformin hydrochloride products (METs). METHODS: Different METs extracted by a dichloromethane/aqueous system were analyzed by HRAM-GC-MS under softer electron ionization (EI) at 30 eV. The accurate masses of NDMA and its internal standard NDMA-d6 were analyzed by full scan and targeted selected ion monitoring modes under 60 000 and 30 000 full width at half maximum at m/z 200, respectively. Data acquisition and processing were managed by Xcalibur and Trace Finder software, respectively. RESULTS: Limits of detection (LOD) and quantification (LOQ) at 10 and 20 ng/g were achieved, which is below the allowed daily intake of 32 ng/g. The mass errors measured from experimental data were within ±2 ppm of the theoretical values over a period of a week. Sample analysis showed that 180 out of 212 samples (85%) were below LOD and 15 out of 212 samples (7 %) were within LOD and LOQ. Only 17 samples (8%) were found to be above LOQ, comprising one active pharmaceutical ingredient (API), five immediate-release METs and 11 extended-released METs. Amongst these, seven extended-release METs and one API exceeded the daily allowed intake, 32 ng/g. CONCLUSIONS: The validated method has been successfully applied for NDMA analysis in various forms of METs. The method is rather straightforward without an additional clean-up step. The scope can also be extended to other volatile impurities in finished pharmaceutical products.

Evaluation of the PIK3 pathway in peripheral T-cell lymphoma and NK/T-cell lymphoma.

Peripheral T-cell lymphomas (PTCL) and natural killer (NK)/T-cell lymphomas (NKTCL) are a heterogeneous group of aggressive malignancies with dismal outcomes and limited treatment options. While the phosphatidylinositol 3-kinase (PIK3) pathway has been shown to be highly activated in many B-cell lymphomas, its therapeutic relevance in PTCL and NKTCL remains unclear. The aim of this study is to investigate the expression of PIK3 and phosphatase and tensin homolog (PTEN) in these subtypes of lymphoma and to identify potential therapeutic targets for clinical testing. Therefore, the expression of PIK3α, PIK3ß, PIK3γ, PIK3δ and PTEN was analyzed in 88 cases of PTCL and NKTCL samples by immunohistochemistry. All PTCL and NKTCL samples demonstrated high expression of PIK3 isoforms. In particular, high PIK3α expression was significantly associated with poor survival, even after adjustment for age, International Prognostic Index (IPI) score and anthracycline-based chemotherapy in first line. Notably, copanlisib, a pan-class I inhibitor with predominant activities towards PIK3α and PIK3δ isoforms, effectively inhibited phosphorylation of AKT, 4E-BP-1 and STAT3, causing G0 /G1 cell cycle arrest and resulting in suppression of tumour cell growth in vitro and in vivo. This study provides evidence that targeting the PIK3 pathway, particularly simultaneous inhibition of PIK3α and δ, could be a promising approach for the treatment of PTCL and NKTCL.

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma.

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

An integrated package for bisulfite DNA methylation data analysis with Indel-sensitive mapping.

BACKGROUND: DNA methylation plays crucial roles in most eukaryotic organisms. Bisulfite sequencing (BS-Seq) is a sequencing approach that provides quantitative cytosine methylation levels in genome-wide scope and single-base resolution. However, genomic variations such as insertions and deletions (indels) affect methylation calling, and the alignment of reads near/across indels becomes inaccurate in the presence of polymorphisms. Hence, the simultaneous detection of DNA methylation and indels is important for exploring the mechanisms of functional regulation in organisms. RESULTS: These problems motivated us to develop the algorithm BatMeth2, which can align BS reads with high accuracy while allowing for variable-length indels with respect to the reference genome. The results from simulated and real bisulfite DNA methylation data demonstrated that our proposed method increases alignment accuracy. Additionally, BatMeth2 can calculate the methylation levels of individual loci, genomic regions or functional regions such as genes/transposable elements. Additional programs were also developed to provide methylation data annotation, visualization, and differentially methylated cytosine/region (DMC/DMR) detection. The whole package provides new tools and will benefit bisulfite data analysis. CONCLUSION: BatMeth2 improves DNA methylation calling, particularly for regions close to indels. It is an autorun package and easy to use. In addition, a DNA methylation visualization program and a differential analysis program are provided in BatMeth2. We believe that BatMeth2 will facilitate the study of the mechanisms of DNA methylation in development and disease. BatMeth2 is an open source software program and is available on GitHub ( https://github.com/GuoliangLi-HZAU/BatMeth2 /).

BatAlign: an incremental method for accurate alignment of sequencing reads.

Structural variations (SVs) play a crucial role in genetic diversity. However, the alignments of reads near/across SVs are made inaccurate by the presence of polymorphisms. BatAlign is an algorithm that integrated two strategies called 'Reverse-Alignment' and 'Deep-Scan' to improve the accuracy of read-alignment. In our experiments, BatAlign was able to obtain the highest F-measures in read-alignments on mismatch-aberrant, indel-aberrant, concordantly/discordantly paired and SV-spanning data sets. On real data, the alignments of BatAlign were able to recover 4.3% more PCR-validated SVs with 73.3% less callings. These suggest BatAlign to be effective in detecting SVs and other polymorphic-variants accurately using high-throughput data. BatAlign is publicly available at https://goo.gl/a6phxB.

Determination of N-nitroso folic acid in folic acid and multivitamin supplements by LC-MS/MS.

Analysis of N-nitroso folic acid, a nitrosamine impurity found in folic acid, is challenging due to the complex sample matrices. Many of such supplements contain not only a variety of vitamins, including vitamin A, Bs, C, D and E, but also other ingredients such as minerals, docosahexaenoic acid (DHA), glucose syrup, sugar, and herbs. On the other hand, the strength of folic acid is typically low, ranging from 50 µg to 5 mg per unit. In this study, a highly selective and sensitive LC-MS/MS method was developed to accurately quantify N-nitroso folic acid in supplements containing folic acid. The sample was extracted by 0.1% ammonia solution: MeOH (9:1, v/v) containing 5 ng/mL of N-nitroso folic acid-d4 (Isotope internal standard). The quantification was performed by MRM in negative ionization mode. Mobile phases A and B were 0.1% formic acid in deionized water and methanol, respectively. The method was validated and found to have sufficient linearity (R2 > 0.995), accuracy (recovery 83-110%), precision (RSD 3%) and low LOD, LOQ (4 and 10 µg/g respectively, with respect to folic acid). The method was applied to the determination of N-nitroso folic acid in 40 supplements containing folic acid with different strengths and formulation. The content of N-nitroso folic acid was found to be up to 898 ng/unit (1794 µg/g with respect to folic acid). It enabled regulatory actions, such as product recall, to safeguard public health from unsafe products.

Aberrant JAK-STAT signaling-mediated chromatin remodeling impairs the sensitivity of NK/T-cell lymphoma to chidamide.

BACKGROUND: Natural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear. METHODS: We performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial. RESULTS: We demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide. CONCLUSIONS: Our study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278.

BatMeth: improved mapper for bisulfite sequencing reads on DNA methylation.

DNA methylation plays a crucial role in higher organisms. Coupling bisulfite treatment with next generation sequencing enables the interrogation of 5-methylcytosine sites in the genome. However, bisulfite conversion introduces mismatches between the reads and the reference genome, which makes mapping of Illumina and SOLiD reads slow and inaccurate. BatMeth is an algorithm that integrates novel Mismatch Counting, List Filtering, Mismatch Stage Filtering and Fast Mapping onto Two Indexes components to improve unique mapping rate, speed and precision. Experimental results show that BatMeth is faster and more accurate than existing tools. BatMeth is freely available at http://code.google.com/p/batmeth/.