Correction to: Molecular signature comprising 11 platelet-genes enables accurate blood-based diagnosis of NSCLC.

An amendment to this paper has been published and can be accessed via the original article.

Molecular signature comprising 11 platelet-genes enables accurate blood-based diagnosis of NSCLC.

BACKGROUND: Early diagnosis is crucial for effective medical management of cancer patients. Tissue biopsy has been widely used for cancer diagnosis, but its invasive nature limits its application, especially when repeated biopsies are needed. Over the past few years, genomic explorations have led to the discovery of various blood-based biomarkers. Tumor Educated Platelets (TEPs) have, of late, generated considerable interest due to their ability to infer tumor existence and subtype accurately. So far, a majority of the studies involving TEPs have offered marker-panels consisting of several hundreds of genes. Profiling large numbers of genes incur a significant cost, impeding its diagnostic adoption. As such, it is important to construct minimalistic molecular signatures comprising a small number of genes. RESULTS: To address the aforesaid challenges, we analyzed publicly available TEP expression profiles and identified a panel of 11 platelet-genes that reliably discriminates between cancer and healthy samples. To validate its efficacy, we chose non-small cell lung cancer (NSCLC), the most prevalent type of lung malignancy. When applied to platelet-gene expression data from a published study, our machine learning model could accurately discriminate between non-metastatic NSCLC cases and healthy samples. We further experimentally validated the panel on an in-house cohort of metastatic NSCLC patients and healthy controls via real-time quantitative Polymerase Chain Reaction (RT-qPCR) (AUC = 0.97). Model performance was boosted significantly after artificial data-augmentation using the EigenSample method (AUC = 0.99). Lastly, we demonstrated the cancer-specificity of the proposed gene-panel by benchmarking it on platelet transcriptomes from patients with Myocardial Infarction (MI). CONCLUSION: We demonstrated an end-to-end bioinformatic plus experimental workflow for identifying a minimal set of TEP associated marker-genes that are predictive of the existence of cancers. We also discussed a strategy for boosting the predictive model performance by artificial augmentation of gene expression data.

Real-Time Molecular Monitoring in Acute Myeloid Leukemia With Circulating Tumor DNA.

The clonal evolution of acute myeloid leukemia (AML), an oligoclonal hematological malignancy, is driven by a plethora of cytogenetic abnormalities, gene mutations, abnormal epigenetic patterns, and aberrant gene expressions. These alterations in the leukemic blasts promote clinically diverse manifestations with common characteristics of high relapse and drug resistance. Defining and real-time monitoring of a personalized panel of these predictive genetic biomarkers is rapidly being adapted in clinical setting for diagnostic, prognostic, and therapeutic decision-making in AML. A major challenge remains the frequency of invasive biopsy procedures that can be routinely performed for monitoring of AML disease progression. Moreover, a single-site biopsy is not representative of the tumor heterogeneity as it is spatially and temporally constrained and necessitates the understanding of longitudinal and spatial subclonal dynamics in AML. Hematopoietic cells are a major contributor to plasma cell-free DNA, which also contain leukemia-specific aberrations as the circulating tumor-derived DNA (ctDNA) fraction. Plasma cell-free DNA analysis holds immense potential as a minimally invasive tool for genomic profiling at diagnosis as well as clonal evolution during AML disease progression. With the technological advances and increasing sensitivity for detection of ctDNA, both genetic and epigenetic aberrations can be qualitatively and quantitatively evaluated. However, challenges remain in validating the utility of liquid biopsy tools in clinics, and universal recommendations are still awaited towards reliable diagnostics and prognostics. Here, we provide an overview on the scope of ctDNA analyses for prognosis, assessment of response to treatment and measurable residual disease, prediction of disease relapse, development of acquired resistance and beyond in AML.

Next generation sequencing guided treatment modulation and prognosis in Acute myeloid leukemia: Case vignettes.

OBJECTIVE: The genomic mutational landscape of Acute Myeloid Leukemia has contributed to better treatment options, risk stratification and prognostication of this genetically heterogeneous disease. With several approved new drugs targeting specific mutations with better outcomes, we describe here two cases of AML in which, NPM1 was detected at diagnosis. The impact of age, type of treatment, stability of NPM1 mutation, and co-occurring mutations on survival are the essential parameters for investigation. METHOD: Both the cases of AML were females, >60 years of age with normal 46XX karyotype. Allele specific RT-PCR and fragment analysis was performed for the detection of NPM1-A mutation at diagnosis. Both the patients were unfit for intensive chemotherapy therefore reduced intensity induction chemotherapy regimen was initially administered. Next-generation sequencing was performed for comprehensive mutational profiling, which guided targeted treatment, prognostic stratification, and response assessment. RESULT: We report that the older AML patients with NPM1 mutation may not have a good outcome with intensive chemotherapy, especially patients with concurrent DNMT3A/IDH-1/2 mutations. In the second case with mutated NPM1, concurrent FLT3-ITD mutation served as a therapeutic target. The FLT3 inhibitor used in combination with standard therapy showed promising results in this case. CONCLUSION: Here, we emphasize on the utility of next generation sequencing in guiding the treatment initiation or modulation during the disease course and risk stratification in AML. In conclusion, conventional chemotherapy in AML gives very poor overall survival rates and targeted chemotherapy against specific mutations may drastically improve patient survival and treatment outcomes.

Rapid Identification of Key Copy Number Alterations in B- and T-Cell Acute Lymphoblastic Leukemia by Digital Multiplex Ligation-Dependent Probe Amplification.

Recurrent clonal genetic alterations are the hallmark of Acute Lymphoblastic Leukemia (ALL) and govern the risk stratification, response to treatment and clinical outcome. In this retrospective study conducted on ALL patient samples, the purpose was to estimate the copy number alterations (CNAs) in ALL by digitalMLPA (dMLPA), validation of the dMLPA data by conventional MLPA and RT-PCR, and correlation of CNAs with Minimal Residual Disease (MRD) status. The ALL patient samples (n = 151; B-ALL, n = 124 cases and T-ALL, n = 27 cases) were assessed for CNAs by dMLPA for detection of sub-microscopic CNAs and ploidy status. This assay allowed detection of ploidy changes and CNAs by multiplexing of karyotyping probes and probes covering 54 key gene targets implicated in ALL. Using the dMLPA assay, CNAs were detected in ~89% (n = 131) of the cases with 66% of the cases harboring ≥3 CNAs. Deletions in CDKN2A/B, IKZF1, and PAX5 genes were detectable in a quarter of these cases. Heterozygous and homozygous gene deletions, and duplications were observed in genes involved in cell cycle control, tumor suppression, lineage differentiation, lymphoid signaling, and transcriptional regulators with implications in treatment response and survival outcome. Distinct CNAs profiles were evident in B-ALL and T-ALL cases. Additionally, the dMLPA assay could reliably identify ploidy status and copy number-based gene fusions (SIL-TAL1, NUP214-ABL, EBF1-PDGFRB). Cases of B-ALL with no detectable recurrent genetic abnormalities could potentially be risk stratified based on the CNA profile. In addition to the commonly used gene deletions for risk assessment (IKZF1, EBF1, CDKN2A/B), we identified a broader spectrum of gene alterations (gains of- RUNX1, LEF1, NR3C2, PAR1, PHF6; deletions of- NF1, SUZ12, MTAP) that significantly correlated with the status of MRD clearance. The CNAs detected by dMLPA were validated by conventional MLPA and showed high concordance (r = 0.99). Our results demonstrated dMLPA to be a robust and reliable alternative for rapid detection of key CNAs in newly diagnosed ALL patients. Integration of ploidy status and CNAs detected by dMLPA with cytogenetic and clinical risk factors holds great potential in further refinement of patient risk stratification and response to treatment in ALL.

Ultrafast spectroscopic study on caffeine mediated dissociation of mutagenic ethidium from synthetic DNA and various cell nuclei.

We report a systematic investigation of caffeine-induced dissociation of ethidium (Et) cation, a potential mutagen. Time-resolved fluorescence studies are consistent with a mechanism where caffeine-Et complex formation in bulk solution drives the dissociation of DNA-bound Et. Temperature-dependent picosecond-resolved studies show the caffeine-Et complex to be stable over a wide range of temperature, within and beyond the normal physiological limit. A combination of NMR spectroscopy and dynamic light scattering experiments allowed us to propose a molecular model of the caffeine-Et complex. Caffeine-induced extraction of Et from whole cells was also performed on squamous epithelial cells collected from the inner lining of the human mouth, A549 (lung carcinoma), A375 (human skin), RAW (macrophage), and Vero (African green monkey kidney epithelium) cell lines. Interestingly the efficiency of caffeine in extracting Et has been found to be dependent on cell types. Our results both in vitro as well as ex vivo provide important clues about the efficiency and mechanism of caffeine as a potential antimutagenic therapeutic agent.

A case of large renal hydatid cyst.

Isolated renal echinococcosis is a rare acquired disease of the kidney, caused by the parasite Echinococcus. The kidneys are involved in less than 2 % of all human hydatidosis; isolated renal involvement is even rarer. Based on the clinical and radiological findings, diagnosis of a rare case of isolated renal echinococcosis was made. The diagnosis was confirmed by laparoscopic surgery and histopathology.

Luminescent quantum clusters of gold in bulk by albumin-induced core etching of nanoparticles: metal ion sensing, metal-enhanced luminescence, and biolabeling.

The synthesis of a luminescent quantum cluster (QC) of gold with a quantum yield of approximately 4 % is reported. It was synthesized in gram quantities by the core etching of mercaptosuccinic acid protected gold nanoparticles by bovine serum albumin (BSA), abbreviated as Au(QC)@BSA. The cluster was characterized and a core of Au(38) was assigned tentatively from mass spectrometric analysis. Luminescence of the QC is exploited as a "turn-off" sensor for Cu(2+) ions and a "turn-on" sensor for glutathione detection. Metal-enhanced luminescence (MEL) of this QC in the presence of silver nanoparticles is demonstrated and a ninefold maximum enhancement is seen. This is the first report of the observation of MEL from QCs. Folic acid conjugated Au(QC)@BSA was found to be internalized to a significant extent by oral carcinoma KB cells through folic acid mediated endocytosis. The inherent luminescence of the internalized Au(QC)@BSA was used in cell imaging.

Bright, NIR-emitting Au23 from Au25: characterization and applications including biolabeling.

A novel interfacial route has been developed for the synthesis of a bright-red-emitting new subnanocluster, Au(23), by the core etching of a widely explored and more stable cluster, Au(25)SG(18) (in which SG is glutathione thiolate). A slight modification of this procedure results in the formation of two other known subnanoclusters, Au(22) and Au(33). Whereas Au(22) and Au(23) are water soluble and brightly fluorescent with quantum yields of 2.5 and 1.3 %, respectively, Au(33) is organic soluble and less fluorescent, with a quantum yield of 0.1 %. Au(23) exhibits quenching of fluorescence selectively in the presence of Cu(2+) ions and it can therefore be used as a metal-ion sensor. Aqueous- to organic-phase transfer of Au(23) has been carried out with fluorescence enhancement. Solvent dependency on the fluorescence of Au(23) before and after phase transfer has been studied extensively and the quantum yield of the cluster varies with the solvent used. The temperature response of Au(23) emission has been demonstrated. The inherent fluorescence of Au(23) was used for imaging human hepatoma cells by employing the avidin-biotin interaction.