Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments–certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing ...screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions ( n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample ...requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.
Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that ...host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.
Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays ...requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.
Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, ...paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory.
We performed an
experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials.
The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (
< 0.001).
We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.
A subset of patients with chronic myelogenous leukemia (CML) do not respond to the tyrosine kinase inhibitor (TKI) imatinib mesylate. Such primary imatinib resistance is distinguished from secondary ...resistance which reemerges after attainment of cytogenetic remission.
We studied gene expression patterns in total WBCs using a panel of 21 genes previously implicated in TKI handling, resistance, or progression comparing patients who had newly diagnosed TKI-naive CML that had optimal (n = 41), or suboptimal (n = 7) responses to imatinib, or primary resistance (n = 20). Expression patterns were compared to those in secondary TKI-resistant chronic phase CML without ABL1 kinase domain mutations (n = 29), and to lymphoid (n = 15) or myeloid blast phase disease (n = 12).
Fifteen genes in the panel distinguished blast phase from chronic phase disease, and 12 genes distinguished newly diagnosed CML from TKI-resistant CML without ABL1 kinase domain mutations, but only a single gene, prostaglandin-endoperoxide synthase 1/cyclooxgenase 1 (PTGS1/COX1; P = .005), differentiated imatinib-responsive from primary imatinib-resistant CML. The association of primary imatinib resistance with higher transcript levels of the drug metabolism gene PTGS1 was confirmed in a separate data set of 68 newly diagnosed, imatinib-treated CML (P = .008). In contrast, up to 11 different genes were identified in a multivariate model that optimally discriminated secondary imatinib resistance lacking ABL1 kinase domain mutation from imatinib-responsive cases, likely related to the more complex pathogenesis of secondary resistance.
Gene expression profiling of CML at diagnosis for PTGS1 may be useful in predicting imatinib response and in selecting alternate therapy.
Fig. Emission spectra of Ho3+ doped PCfBfTiH glasses upon 451 nm excitation.
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•Ho3+-doped oxyfluoride glasses have been synthesized by conventional melt-quenching technique.•Thermal, ...optical and photoluminescence properties Ho3+:glasses have been performed.•Intense green and red emissions were obtained upon excitation of 451 nm.•Decay curves of the 5S2 + 5F4 → 5I8 transition of Ho3+ ions have been analyzed by double exponential function.
Holmium (Ho3+)-doped fluorophosphate glasses (P2O5–BaF2–CaF2–TiO2–Ho2O3, PCfBfTiH) were fabricated by melt-quenching technique. The glass transition (548 °C) and crystallization (577 °C) temperatures and glass stability factor (161 °C) were obtained from DSC analysis. Judd-Ofelt (JO) intensity parameters (Ω2, Ω4 and Ω6) of PCfBfTiH15 glass were evaluated. Intense green (545 nm) emission was obtained for the 5S2 + 5F4 → 5I8 transition upon excitation of 451 nm. The stimulated emission cross-section (σem) of PCfBfTiH15 glass was found to be as high as 2.01 × 10−20 cm2. Decay curves of the 5S2 + 5F4 → 5I8 transition were analyzed by double exponential function. High lifetime of 11.29 μs for the 5S2 + 5F4 level was obtained. The CIE chromaticity coordinates for the emission spectra were professed that the green emission owing to the 5S2 + 5F4 → 5I8 transition may be useful for laser and display applications at around 545 nm.
Gain cross-section spectra of Er3+-doped PCfBfTiEr20 glass for the 4I13/2 → 4I15/2 transition.
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•Erbium (Er3+)-doped oxyfluoride (PCfBfTiEr) glasses have been synthesized.•Judd-Ofelt ...(JO) phenomenological intensity parameters (Ω2, Ω4, Ω6) have been estimated.•Intense eye-safe emission at 1534 nm has been obtained under 980 nm laser excitation.•High emission cross-section and bandwidth product were obtained for PCfBfTiEr2.0 glass.
Optical and visible upconversion properties of erbium (Er3+)-doped oxyfluoro-titania-phosphate glasses (PCfBfTiEr) with the chemical composition of P2O5–CaF2–BaF2–TiO2–Er2O3 have been explored. An intense emission at 1.53 µm of Er3+-doped PCfBfTiEr1.0 glass was obtained upon excitation of 980 nm diode laser. In addition, green and red visible upconversion emissions were obtained upon the optical excitation of Er3+ ions doped PCfBfTiEr glasses at 980 nm diode laser. Upconversion emission intensities and population densities of respective levels were tuned with the variation of Er3+ ion concentration. Fluorescence decay curves of the 4I13/2 level of PCfBfTiEr glasses were obtained upon 980 nm laser excitation in the pulsed mode and revealed a mono-exponential behavior. The stimulated emission cross-section (σem), full width at half maximum (FWHM) and gain bandwidth product (σem × FWHM) were found to be 9.3 × 10−21 cm2, 95.61 nm and 889.2 cm−2 nm for PCfBfTiEr2.0 glass, respectively. These results recommend that the Er3+ ions doped PCfBfTiEr glasses may possibly be worthy for the laser and optical amplification applications at 1.53 µm.
Pandemic is a well-known term for the year 2020. It's essentially a disease that spreads across a region or the entire planet. The entire planet appears powerless, and a jerk triggered by a virus ...outbreak has halted. On 11 March 2020, WHO announced Corona Virus disease 2019 (COVID-19) a pandemic. The outbreak or epidemic of the virus differs widely from one nation to another. Society is the secret to solving the pandemic. Fever is one of the common, easily detectable symptoms of COVID-19. The COVID 19 in India is one of the most widespread pandemics caused by extreme acute corona viral syndrome2 (SARS-CoV-2) in coronvirus disease 2019 (COVID-19). On 30 January 2020, the original case of COVID-19 in India, arising in China, was registered. India currently has the highest number of confirmed cases in Asia and the second largest number after the United States of America in the world, with a combined number of confirmed cases exceeding the thresholds of 100,000 on 19 May and 1.000,000 confirmed cases on 17 July 2020. The largest one-day increase in COVID-19 cases of 78,761 cases was observed in Indian countries on 29 August 2020, surpassing the previous record in US cases of 77,368 on 17 July 2020. Nowadays, data science tasks are not limited to traditional data analysis with limited attributes and records. In current scenarios, the real-time datasets are huge with enormous attributes and such datasets are very complex to evaluate using classical data analysis tools. For example, the datasets of the medical domain integrate several attributes in which the information of symptoms, diagnosis, travel history, health parameters, and many others are evaluated. To deal with such types of datasets assorted database query tools and programming languages are used.
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