Genome-Wide Comparison of Next-Generation Sequencing and qPCR Platforms for microRNA Profiling in Serum

Blöndal, Thórarinn; Brunetto, Maurizia Rossana; Cavallone, D.; Mikkelsen, Martin; Thorsen, Michael; Mang, Yuan; Pinheiro, Hazel; Bonino, Ferruccio; Mouritzen, Peter

doi:10.1007/978-1-4939-6866-4_3

Cited by 17 publications

(19 citation statements)

References 12 publications

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“…More recently, miR-4734 and miR-150-5p have been associated with prognosis in early breast cancer patients treated with adjuvant trastuzumab (43). These conflicting results can be ascribed to moderately sized patient cohorts, heterogeneity of disease stage or treatment type, different sample source (plasma or serum), profiling platforms, normalization approaches, and limited number of ct-miRNAs analyzed (44)(45)(46)(47). Our ct-miRNA signatures were built on high-throughput analysis of samples collected within a controlled randomized study and were developed through a statistical strategy based on penalized regression model, which controls overfitting and is based on the principle of parsimony (40,44).…”

Section: Discussionmentioning

confidence: 99%

Plasma miRNA Levels for Predicting Therapeutic Response to Neoadjuvant Treatment in HER2-positive Breast Cancer: Results from the NeoALTTO Trial

Cosimo

Appierto

Pizzamiglio

et al. 2019

Clinical Cancer Research

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Purpose: To investigate the potential of circulating-miRNAs (ct-miRNA) as noninvasive biomarkers to predict the efficacy of single/dual HER2-targeted therapy in the NeoALTTO study. Experimental Design: Patients with plasma samples at baseline (T0) and/or after 2 weeks (T1) of treatment were randomized into training (n ¼ 183) and testing (n ¼ 246) sets. RT-PCR-based high-throughput miRNA profiling was employed in the training set. After normalization, ct-miRNAs associated with pathologic complete response (pCR) were identified by univariate analysis. Multivariate logistic regression models were implemented to generate treatment-specific signatures at T0 and T1, which were evaluated by RT-PCR in the testing set. Event-free survival (EFS) according to ct-miRNA signatures was estimated by Kaplan-Meier method and Cox regression model. Results: In the training set, starting from 51 ct-miRNAs associated with pCR, six signatures with statistically significant predictive capability in terms of area under the ROC curve (AUC) were identified. Four signatures were confirmed in the testing set: lapatinib at T0 and T1 [AUC 0.86; 95% confidence interval (CI), 0.73-0.98 and 0.71 (0.55-0.86)], respectively; trastuzumab at T1 (0.81; 0.70-0.92); lapatinib þ trastuzumab at T1 (0.67; 0.51-0.83). These signatures were confirmed predictive after adjusting for known variables, including estrogen receptor status. ct-miRNA signatures failed to correlate with EFS. However, the levels of ct-miR-140-5p, included in the trastuzumab signature, were associated with EFS (HR 0.43; 95% CI, 0.22-0.84). Conclusions: ct-miRNAs discriminate patients with and without pCR after neoadjuvant lapatinib-and/or trastuzumab-based therapy. ct-miRNAs at week two could be valuable to identify patients responsive to trastuzumab, to avoid unnecessary combination with other anti-HER2 agents, and finally to assist deescalating treatment strategies.

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Section: Discussionmentioning

confidence: 99%

Plasma miRNA Levels for Predicting Therapeutic Response to Neoadjuvant Treatment in HER2-positive Breast Cancer: Results from the NeoALTTO Trial

Cosimo

Appierto

Pizzamiglio

et al. 2019

Clinical Cancer Research

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“…Ultimately, we validated the mass cytometry results by translating and applying the main findings into smaller flow cytometry panels. Since mass cytometry is still a relatively new method, validation by other methods is vital, as is the norm for new techniques (41). …”

Section: Introductionmentioning

confidence: 99%

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

et al. 2017

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Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets.

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“…The availability of powerful approaches for global miRNA characterization such as microarrays [97], NGS [98][99][100][101][102][103][104][105][106] and simple, universally applicable assays for quantification of miRNA expression such as qPCR [107] and statistical/bioinformatics methods for data analyses and interpretation [108][109][110], suggests that the validation pipeline that often encounters bottlenecks [17] will be more efficient in this assay. There is a pressing need for accelerating use of sensitive and stable molecular markers, such as miRNA molecules, in non-or minimally-invasive media such as stool and/or blood to improve the detection of CRC [111], particularly at an early tumor lymph node metastasis (TNM) disease stage (0-1) [112,113] while the cancer is still curable.…”

Section: Current Methods For Colon Cancer Screeningmentioning

confidence: 99%

“…A scatter plot comparing low dose microarray data to the control group, and data presented in figure 3 shows a multigroup plot comparing miRNA-193a-3p to internal standard 18S rRNA in healthy normal control and the four TNM colon cancer groups (stages 0 to IV). Table 4 presents comparison of NGS with qPCR technologies for miRNA profiling [98][99][100][101][102][103][104]. …”

Section: Microarray Technologiesmentioning

confidence: 99%

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

Ahmed¹,

Ahmed²

2017

Med Res Innov

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Screening for colon cancer (CC) allows for diagnosis of the early stage for malignancy and potentially reduces disease mortality as the cancer could be cured at disease earliest stages. Early detection could be desirable if accurate, practical and cost effective diagnostic measures for this cancer are available. Mortality and morbidity from colon cancer represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lacks sensitivity and requires dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could lead to mortality. In comparison to the FOBT test, a noninvasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer (CRC) is the only cancer for which screening by colonoscopy is recommended. Although colonoscopy is a reliable screening tool, its invasive nature, accompanying abdominal pain, potential complications and high cost have hampered the application of this screening method worldwide.A novel screening approach using the stable miRNA molecules, which are relatively nondegradable when extracted from noninvasive stool and semi-invasive blood samples by currently available commercial kits and manipulated thereafter, would be preferable to a transcriptomic messenger (m)RNA-, a mutation DNA-, an epigenetic-or a proteomic-based test. The approach utilizes reverse transcriptase (RT), followed by a modified quantitative real-time polymerase chain reaction (qPCR). Although exosomal RNA would not be measured, using a restricted extraction of total RNA from stool or blood, then a parallel test could also be carried out on RNA obtained from stool or plasma samples, and appropriate corrections for exsosomal loss can be made to obtain accurate and quantitative results. Eventually, a chip would be developed to facilitate diagnosis, as has been carried out for the quantification of genetically modified organisms (GMOs) in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy. If performance criteria are met, as detailed herein, then a miRNA test in human stool or blood samples based on high throughput automated technologies and quantitative expression measurements --commonly used in the diagnostic clinical laboratory--would eventually be advanced to the clinical setting, which will make a vivid impact on the prevention of colon cancer.Correspondence to: Farid E. Ahmed, GEM

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Genome-Wide Comparison of Next-Generation Sequencing and qPCR Platforms for microRNA Profiling in Serum

Cited by 17 publications

References 12 publications

Plasma miRNA Levels for Predicting Therapeutic Response to Neoadjuvant Treatment in HER2-positive Breast Cancer: Results from the NeoALTTO Trial

Plasma miRNA Levels for Predicting Therapeutic Response to Neoadjuvant Treatment in HER2-positive Breast Cancer: Results from the NeoALTTO Trial

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

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