The diagnostic and therapeutic prospects of exosomes in ovarian cancer.

Exosomes are nano-sized vesicles derived from the endosomal system and are involved in many biological and pathological processes. Emerging evidence has demonstrated that exosomes with cell-specific constituents are associated with the tumorigenesis and progression of ovarian cancer. Therefore, exosomes derived from ovarian cancers can be potential diagnostic biomarkers and therapeutic targets. In this review, we briefly present the biological characteristics of exosomes and the recent advances in isolating and detecting exosomes. Furthermore, we summarise the many functions of exosomes in ovarian cancer, hoping to provide a theoretical basis for clinical applications of exosomes in the diagnosis and treatment of ovarian cancer.

Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis.

The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers.

Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

BACKGROUND: To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. METHODS: Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. RESULTS: Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. CONCLUSION: We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

What Are the Biomarkers for Immunotherapy in SCLC?

Small-cell lung cancer (SCLC) is an aggressive malignancy that exhibits a rapid doubling time, a high growth fraction, and the early development of widespread metastases. The addition of immune checkpoint inhibitors to first-line chemotherapy represents the first significant improvement of systemic therapy in several decades. However, in contrast to its effects on non-SCLC, the advantageous effects of immunotherapy addition are modest in SCLC. In particular, only a small number of SCLC patients benefit from immune checkpoint inhibitors. Additionally, biomarkers selection is lacking for SCLC, with clinical trials largely focusing on unselected populations. Here, we review the data concerning the major biomarkers for immunotherapy, namely, programmed death ligand 1 expression and tumour mutational burden. Furthermore, we explore other potential biomarkers, including the role of the immune microenvironment in SCLC, the role of genetic alterations, and the potential links between neurological paraneoplastic syndromes, serum anti-neuronal nuclear antibodies, and outcomes in SCLC patients treated with immunotherapy.

Evaluating the impact of age on immune checkpoint therapy biomarkers.

Both tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age.

Absolute quantification of 2-hydroxyglutarate on tissue by matrix-assisted laser desorption/ionization mass spectrometry imaging for rapid and precise identification of isocitrate dehydrogenase mutations in human glioma.

Isocitrate dehydrogenase (IDH) gene mutations are important predictive molecular markers to guide surgical strategy in brain cancer therapy. Herein, we presented a method using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for absolute quantification of 2-hydroxyglutarate (2-HG) on tissues to identify IDH mutations and evaluate tumor residue. This analytical method was tested among 34 glioma patients and validated with gold standard clinical technologies. The cut-off value of 2-HG was set as 0.81 pmol/µg to identify IDH mutant (IDHmt) gliomas with 100% specificity and sensitivity. In addition, 2-HG levels and tumor cell density (TCD) showed positive correlation in IDHmt gliomas by this spatial method. This MALDI MSI-based absolute quantification method has great potentiality for incorporating into surgical workflow in the future.

Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues.

To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and α = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available via ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age (p < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn® SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis (p > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.

High-throughput screening of salivary polyamine markers for discrimination of colorectal cancer by multisegment injection capillary electrophoresis tandem mass spectrometry.

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.

TEP linc-GTF2H2-1, RP3-466P17.2, and lnc-ST8SIA4-12 as novel biomarkers for lung cancer diagnosis and progression prediction.

PURPOSE: Platelets contain a rich repertoire of RNA species, such as mRNAs and long non-coding RNAs. During the development of tumors, platelets are "educated" by cancer cells, altering their transcriptome and molecular content, thereby, tumor educated platelet (TEP) lncRNA profile has the potential to diagnose lung cancer. The current study was aimed to examine whether TEPs might be a potential biomarker for lung cancer. METHODS: Platelet precipitation was obtained by low-speed centrifugation. TEP linc-GTF2H2-1, RP3-466P17.2, and lnc-ST8SIA4-12 were selected by lncRNA microarray and validated by qPCR in a large cohort of lung cancer patients and healthy donors. Besides, we analyzed the association of their expression levels with clinicopathological features. RESULTS: TEP linc-GTF2H2-1 and RP3-466P17.2 were significantly downregulated, while lnc-ST8SIA4-12 was significantly upregulated in patients with lung cancer or with early-stage lung cancer as compared to healthy donors, possessing AUCs of 0.781, 0.788, 0.725 for lung cancer and 0.704, 0.771, 0.768 for early-stage lung cancer, respectively. Notably, their combination demonstrated the markedly elevated AUCs of 0.921 for lung cancer and 0.895 for early-stage lung cancer. Besides, the combination of TEP linc-GTF2H2-1 was capable to facilitate diagnostic efficiencies of CEA, Cyfra21-1, or NSE to distinguish advanced-stage lung cancer patients from early ones, with an AUC of 0.899 based on the integration of these four factors. CONCLUSION: Our data suggested that lncRNAs sequestered in TEPs enabled blood-based lung cancer diagnosis and progression prediction.

Visualization of platelet recruitment to tumor lesions using highly sensitive and stable radioiodine studded gold nanoprobes.

In vivo imaging of platelets will provide a better understanding of their critical roles in arterial cardiovascular disease, hemostasis, inflammation, and cancer. Here, we demonstrate the feasibility of using radioiodine studded gold nanoprobes (RIS-GNPs) as a platelet tracker for nuclear medicine imaging in tumor-bearing mice using positron emission tomography and computed tomography (PET/CT). Platelet labeling with RIS-GNPs did not alter the platelet functions, such as cellular proliferation and aggregation. PET/CT imaging clearly revealed the migration of platelets into tumor sites at 1 to 5 h post-transfer of RIS-GNP-labeled platelets, which was consistent with the biodistribution data. Our findings suggest that the imaging approach using RIS-GNPs makes it feasible to visualize the biological behavior of platelets in living organisms with cancer.

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

An integrated workflow for biomarker development using microRNAs in extracellular vesicles for cancer precision medicine.

EV-miRNAs are microRNA (miRNA) molecules encapsulated in extracellular vesicles (EVs), which play crucial roles in tumor pathogenesis, progression, and metastasis. Recent studies about EV-miRNAs have gained novel insights into cancer biology and have demonstrated a great potential to develop novel liquid biopsy assays for various applications. Notably, compared to conventional liquid biomarkers, EV-miRNAs are more advantageous in representing host-cell molecular architecture and exhibiting higher stability and specificity. Despite various available techniques for EV-miRNA separation, concentration, profiling, and data analysis, a standardized approach for EV-miRNA biomarker development is yet lacking. In this review, we performed a substantial literature review and distilled an integrated workflow encompassing important steps for EV-miRNA biomarker development, including sample collection and EV isolation, EV-miRNA extraction and quantification, high-throughput data preprocessing, biomarker prioritization and model construction, functional analysis, as well as validation. With the rapid growth of "big data", we highlight the importance of efficient mining of high-throughput data for the discovery of EV-miRNA biomarkers and integrating multiple independent datasets for in silico and experimental validations to increase the robustness and reproducibility. Furthermore, as an efficient strategy in systems biology, network inference provides insights into the regulatory mechanisms and can be used to select functionally important EV-miRNAs to refine the biomarker candidates. Despite the encouraging development in the field, a number of challenges still hinder the clinical translation. We finally summarize several common challenges in various biomarker studies and discuss potential opportunities emerging in the related fields.

In search for biomarkers and potential drug targets for uterine serous endometrial cancer.

OBJECTIVE: Serous endometrial cancer (USC) is a challenging malignancy associated with metastasis, recurrence and poor outcome. To identify clinically relevant prognostic biomarkers, we focused on a panel of proteins selected after a comprehensive literature review, for tumour profiling of a homogeneous cohort of USC patients. METHODS: Protein levels and localization were assessed by immunohistochemistry analysis in 36 hysterectomy samples. Tissue sections were stained with the following antibodies: Aurora A, phospho (T288) Aurora A, BRCA1, CHK1, CIP2A, Cyclin B1, Cyclin E, E2F-1, phospho (S364) E2F-1, FBXW7, FOXM1, phospho (S9) GSK3Beta, PLK1, phospho (T210) PLK1, PPP2R1B, p73, RAD51. Each marker was evaluated as a continuously-scaled variable for association with disease progression and death, using Cox proportional hazards models. The sample consisted of 36 patients with USC, half with stage III or IV disease. RESULTS: Results showed that higher CHK1 (Checkpoint kinase 1) expression was associated with a decreased risk of progression and death, after adjusting for stage. Interestingly, analysis of a TCGA data set of 109 USC patients corroborates our results showing a favourable prognostic role of CHEK1 after adjusting for stage. Higher FBXW7 (F-box and WD repeat domain containing 7) expression and higher cytoplasmic expression of PPP2R1B (Protein Phosphatase 2 A, Scaffold Subunit Abeta) were each associated with a decreased risk of progression, after adjusting for stage. CONCLUSIONS: In conclusion, results from the present study identify new clinically relevant biomarkers and potential drug targets for uterine serous endometrial cancer.

Quantification of biomarker functionality predicts patient outcomes.

Implementation of a quantitative molecular imaging method (iFRET), which determines receptor-ligand interactions, has led to the finding that patients with a low extent of PD-1/PD-L1 interaction in metastatic NSCLC, and malignant melanoma, display significantly worsened overall survival compared to those with a high level of interaction.

Urinary Cell-Free DNA Integrity Analysis.

Urine cell-free DNA is an important source of diagnostic markers for different diseases, especially for cancer. It could be important to achieve the urine cell-free DNA integrity to establish its provenience from cancer cells or dead inflammatory cells for necrosis in urine or from normal cells with the purpose to use it as an early diagnostic tool for urological cancers or other diseases. Here we describe a simple, noninvasive approach from urine collection to DNA integrity analysis using real-time PCR.

Mutational Analysis in Urinary Cell-Free DNA: KRAS in Colorectal Cancer.

Urinary cell-free DNA offers an important noninvasive source of material for genomic testing also for nonurological tumors. Its clinical utility in monitoring tumor evolution and treatment failure is promising. Here we describe a method to detect cancer mutations into urine from patients affected by colorectal cancer.

Pantoea agglomerans como marcador de cáncer en pacientes inmunodeprimidos / Pantoea agglomerans as a cancer hallmark in immunocompromised patients

Pantoea agglomerans es un bacilo gramnegativo reportado principalmente en heridas penetrantes, infecciones neonatales o como contaminante. La infección relevante por esta bacteria es rara. Nuestra revisión de la literatura sugirió una asociación consistente entre la infección por Pantoea agglomerans en pacientes inmunodeprimidos y una neoplasia maligna previamente diagnosticada. Los autores describen un derrame pleural paraneumónico en el que el aislamiento de Pantoea agglomerans permitió el diagnóstico de novo de cáncer de pulmón

Pantoea agglomerans is a gram-negative rod reported mainly in penetrating wounds, neonatal infections or as a contaminant. Relevant infection by this bacterium is rare. Our review of available literature suggested a consistent association between Pantoea agglomerans infection in immunocompromised patients and previously diagnosed malignancy. The authors describe a parapneumonic pleural effusion in which the isolation of Pantoea agglomerans allowed for de novo diagnosis of lung cancer

Identification of oral squamous cell carcinoma markers MUC2 and SPRR1B downstream of TANGO.

PURPOSE: Transport and Golgi organization protein 1 (TANGO) promotes angiogenesis and lymphangiogenesis in oral squamous cell carcinoma (OSCC). To elucidate the underlying mechanisms, this study aims to identify and characterize elements downstream of TANGO that mediate its involvement in OSCC. METHODS: In this study, microarray analysis compared gene expression between control and TANGO-repressed HSC3 cells. Protein expression in 213 OSCC tissue samples was analyzed immunohistochemically. RESULTS: TANGO repression decreased or increased expression of Mucin 20 (MUC20) and small proline-rich protein 1B (SPRR1B), respectively. MUC20 increased the growth and invasiveness of OSCC cells via altered matrix metalloproteinase (MMP)-2 and E-cadherin expression and c-met phosphorylation. MUC20 induced angiogenesis and lymphangiogenesis by activating vascular endothelial growth factors A and C. In well-differentiated OSCC, SPRR1B expression was high (P = 0.0091) and correlated with keratinization markers and promoted proliferation by inducing mitogen-activated protein kinase p38 phosphorylation. MUC20 expression correlated significantly with clinical stage (P = 0.0024), lymph node metastasis (P = 0.0036), and number of blood and lymph vessels (P < 0.0001). MUC20-expressing cases had a significantly worse prognosis than non-expressing cases (P < 0.0001). CONCLUSION: MUC20 and SPRR1B located downstream of TANGO may be useful molecular markers for OSCC.

Cell-free tumour DNA analysis detects copy number alterations in gastro-oesophageal cancer patients.

BACKGROUND: Analysis of cell-free tumour DNA, a liquid biopsy, is a promising biomarker for cancer. We have performed a proof-of principle study to test the applicability in the clinical setting, analysing copy number alterations (CNAs) in plasma and tumour tissue from 44 patients with gastro-oesophageal cancer. METHODS: DNA was isolated from blood plasma and a tissue sample from each patient. Array-CGH was applied to the tissue DNA. The cell-free plasma DNA was sequenced by low-coverage whole-genome sequencing using a clinical pipeline for non-invasive prenatal testing. WISECONDOR and ichorCNA, two bioinformatic tools, were used to process the output data and were compared to each other. RESULTS: Cancer-associated CNAs could be seen in 59% (26/44) of the tissue biopsies. In the plasma samples, a targeted approach analysing 61 regions of special interest in gastro-oesophageal cancer detected cancer-associated CNAs with a z-score >5 in 11 patients. Broadening the analysis to a whole-genome view, 17/44 patients (39%) had cancer-associated CNAs using WISECONDOR and 13 (30%) using ichorCNA. Of the 26 patients with tissue-verified cancer-associated CNAs, 14 (54%) had corresponding CNAs in plasma. Potentially clinically actionable amplifications overlapping the genes VEGFA, EGFR and FGFR2 were detected in the plasma from three patients. CONCLUSIONS: We conclude that low-coverage whole-genome sequencing without prior knowledge of the tumour alterations could become a useful tool for cell-free tumour DNA analysis of total CNAs in plasma from patients with gastro-oesophageal cancer.

Multiomics Characterization of Low-Grade Serous Ovarian Carcinoma Identifies Potential Biomarkers of MEK Inhibitor Sensitivity and Therapeutic Vulnerability.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.