Single-cell genomics is an approach to investigate cell heterogeneity and to identify new molecular features correlated with clinical outcomes. This approach allows identification of the complexity ...of cell diversity in a sample without the loss of information that occurs when multicellular or bulk tissue samples are analyzed.
The first single-cell RNA-sequencing study was published in 2009, and since then many more studies and single-cell sequencing methods have been published. These studies have had a major impact on several fields, including microbiology, neurobiology, cancer, and developmental biology. Recently, improvements in reliability and the development of commercial single-cell isolation platforms are opening the potential of this technology to the clinical laboratory.
In this review we provide an overview of the current state of single-cell genomics. We describe opportunities in clinical research and medical applications.
Background
Alzheimer's disease (AD) is one of the most common causes of dementia in old people. Neuronal deficits such as loss of memory, language and problem-solving are severely compromised in ...affected patients. The molecular features of AD are Aβ deposits in plaques or in oligomeric structures and neurofibrillary tau tangles in brain. However, the challenge is that Aβ is only one piece of the puzzle, and recent findings continue to support the hypothesis that their presence is not sufficient to predict decline along the AD outcome. In this regard, metabolomic-based techniques are acquiring a growing interest for either the early diagnosis of diseases or the therapy monitoring. Mass spectrometry is one the most common analytical platforms used for detection, quantification, and characterization of metabolic biomarkers. In the past years, both targeted and untargeted strategies have been applied to identify possible interesting compounds.
Aim of review
The overall goal of this review is to guide the reader through the most recent studies in which LC–MS-based metabolomics has been proposed as a powerful tool for the identification of new diagnostic biomarkers in AD. To this aim, herein studies spanning the period 2009–2020 have been reported. Advantages and disadvantages of targeted vs untargeted metabolomic approaches have been outlined and critically discussed.
Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (
) gene mutations at the ...single circulating tumor cell (CTC) level in metastatic breast cancer (MBC).
We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in
and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing.
Among 3 selected patients, 2 had an
mutation (Y537). One showed two different
variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in
(Y537S and T570I), which has not been reported previously.
CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of
mutations, the emergence of endocrine resistance and the choice of further target therapy.
.
Alzheimer's disease (AD), a neurodegenerative disorder, is the most common cause of dementia in the elderly population. Since its original description, there has been intense debate regarding the ...factors that trigger its pathology. It is becoming apparent that AD is more than a brain disease and harms the whole-body metabolism. We analyzed 630 polar and apolar metabolites in the blood of 20 patients with AD and 20 healthy individuals, to determine whether the composition of plasma metabolites could offer additional indicators to evaluate any alterations in the metabolic pathways related to the illness. Multivariate statistical analysis showed that there were at least 25 significantly dysregulated metabolites in patients with AD compared with the controls. Two membrane lipid components, glycerophospholipids and ceramide, were upregulated, whereas glutamic acid, other phospholipids, and sphingolipids were downregulated. The data were analyzed using metabolite set enrichment analysis and pathway analysis using the KEGG library. The results showed that at least five pathways involved in the metabolism of polar compounds were dysregulated in patients with AD. Conversely, the lipid pathways did not show significant alterations. These results support the possibility of using metabolome analysis to understand alterations in the metabolic pathways related to AD pathophysiology.
Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical ...biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.
The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) ...have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of
and
were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous
R248W mutation from one single CTC and pools of three CTCs, and homozygous
R248W mutation from one single CTC and pools of two CTCs. Wild-type
was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
High-grade serous ovarian cancer (HGSOC) patients carrying the
mutation or deficient in the homologous recombination repair system (HRD) generally benefit from treatment with PARP inhibitors. Some ...international recommendations suggest that
genetic testing should be offered for all newly diagnosed epithelial ovarian cancer, along with HRD assessment. Academic tests (ATs) are continuously under development, in order to break down the barriers patients encounter in accessing HRD testing. Two different methods for shallow whole-genome sequencing (sWGS) were compared to the reference assay, Myriad. All these three assays were performed on 20 retrospective HGSOC samples. Moreover, HRD results were correlated with the progression-free survival rate (PFS). Both sWGS chemistries showed good correlation with each other and a complete agreement, even when compared to the Myriad score. Our academic HRD assay categorized patients as HRD-Deficient, HRM-Mild and HRN-Negative. These three groups were matched with PFS, providing interesting findings in terms of HRD scoring and months of survival. Both our sWGS assays and the Myriad test correlated with the patient's response to treatments. Finally, our AT confirms its capability of determining HRD status, with the advantage of being faster, cheaper, and easier to carry out. Our results showed a prognostic value for the HRD score.
Pathogenic variants (PVs) carriers in
or
are associated with an elevated lifetime risk of developing breast cancer (BC) and/or ovarian cancer (OC). The prevalence of
and
germline alterations is ...extremely variable among different ethnic groups. Particularly, the rate of variants in Italian BC and/or OC families is rather controversial and ranges from 8% to 37%, according to different reports. By In Vitro Diagnostic (IVD) next generation sequencing (NGS)-based pipelines, we routinely screened thousands of patients with either sporadic or cancer family history. By NGS, we identified new PVs and some variants of uncertain significance (VUS) which were also evaluated in silico using dedicated tools. We report in detail data regarding
variants identified in 517 out of 2351 BC and OC patients. The aim of this study was to report the incidence and spectrum of
variants observed in BC and/or OC patients, tested in at Policlinico Gemelli Foundation Hospital, the origin of which is mainly from Central and Southern Italy. This study provides an overview of the variant frequency in these geographic areas of Italy and provides data that could be used in the clinical management of patients.
The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral ...capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (
and
) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools.
: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (
,
,
and
) in twenty COVID-19-positive patients' swabs. The
and
were assayed by real-time RT-qPCR and digital PCR.
: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients' swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The
RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while
was within all of the Ct ranges.
: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the
transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.
Alzheimer's disease (AD) is a devastating neurodegenerative disease without guidelines for early diagnosis or personalized treatment. Previous studies have highlighted a crucial role of increasing ...phosphorylation levels of the amyloid precursor protein (APP) Tyr682 residue in predicting neuronal deficits in AD patients. However, the lack of a method for the identification and quantification of Tyr682 phosphorylation levels prevents its potential clinical applications.
Here we report a method to identify and quantify APP Tyr682 phosphorylation levels in blood mononuclear cells of AD patients by tandem mass spectrometry (tMS).
This method showed excellent sensitivity with detection and quantification limits set respectively at 0.035 and 0.082 ng injected for the phosphorylated peptide and at 0.02 and 0.215 ng injected for the non-phosphorylated peptide. The average levels of both peptides were quantified in transfected HELA cells (2.48 and 3.53 ng/μg of protein, respectively). Preliminary data on 3 AD patients showed quantifiable levels of phosphorylated peptide (0.10-0.15 ng/μg of protein) and below the LOQ level of non-phosphorylated peptide (0.13 ng/μg of protein).
This method could allow the identification of patients with increased APP Tyr682 phosphorylation and allow early characterization of molecular changes prior to the appearance of clinical signs.
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