Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Molecular-Based Immunohistochemical Algorithm for Uterine Leiomyosarcoma Diagnosis.

The morphologic assessment of uterine leiomyosarcoma (LMS) may be challenging, and diagnostic immunohistochemical (IHC) analysis is currently lacking. We evaluated the genomic landscape of 167 uterine LMS by targeted next-generation sequencing (NGS) to identify common genomic alterations. IHC analyses corresponding to these genomic landmarks were applied to a test cohort of 16 uterine LMS, 6 smooth muscle tumors of uncertain malignant potential (STUMP), and 6 leiomyomas with NGS data and a validation cohort of 8 uterine LMS, 12 STUMP, 21 leiomyomas and leiomyoma variants, 7 low-grade endometrial stromal sarcomas, and 2 diagnostically challenging uterine smooth muscle tumors. IHC results were individually interpreted by 3 pathologists blinded to NGS data. Overall, 94% of LMS showed ≥1 genomic alteration involving TP53, RB1, ATRX, PTEN, CDKN2A, or MDM2, with 80% showing alterations in ≥2 of these genes. In the test cohort, an initial panel of p53, Rb, PTEN, and ATRX was applied, followed by a panel of DAXX, MTAP, and MDM2 in cases without abnormalities. Abnormal p53, Rb, PTEN, and ATRX IHC expression was seen in 75%, 88%, 44%, and 38% of LMS, respectively, in the test cohort. Two or more abnormal IHC results among these markers were seen in 81% of LMS. STUMPs demonstrated only 1 IHC abnormality involving these markers. No IHC abnormalities were seen in leiomyomas. In the validation cohort, abnormal p53, Rb, and PTEN IHC results were seen in LMS, whereas rare STUMP or leiomyomas with bizarre nuclei showed IHC abnormalities involving only 1 of the markers. Abnormalities in ≥2 markers were present in both diagnostically challenging smooth muscle tumors, confirming LMS. Concordance was excellent among pathologists in the interpretation of IHC (κ = 0.97) and between IHC and NGS results (κ = 0.941). Uterine LMS exhibit genomic landmark alterations for which IHC surrogates exist, and a diagnostic algorithm involving molecular-based IHC may aid in the evaluation of unusual uterine smooth muscle tumors.

Using Comorbidity Pattern Analysis to Detect Reliable Methylated Genes in Colorectal Cancer Verified by Stool DNA Test.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide in 2020. Colonoscopy and the fecal immunochemical test (FIT) are commonly used as CRC screening tests, but both types of tests possess different limitations. Recently, liquid biopsy-based DNA methylation test has become a powerful tool for cancer screening, and the detection of abnormal DNA methylation in stool specimens is considered as an effective approach for CRC screening. The aim of this study was to develop a novel approach in biomarker selection based on integrating primary biomarkers from genome-wide methylation profiles and secondary biomarkers from CRC comorbidity analytics. A total of 125 differential methylated probes (DMPs) were identified as primary biomarkers from 352 genome-wide methylation profiles. Among them, 51 biomarkers, including 48 hypermethylated DMPs and 3 hypomethylated DMPs, were considered as suitable DMP candidates for CRC screening tests. After comparing with commercial kits, three genes (ADHFE1, SDC2, and PPP2R5C) were selected as candidate epigenetic biomarkers for CRC screening tests. Methylation levels of these three biomarkers were significantly higher for patients with CRC than normal subjects. The sensitivity and specificity of integrating methylated ADHFE1, SDC2, and PPP2R5C for CRC detection achieved 84.6% and 92.3%, respectively. Through an integrated approach using genome-wide DNA methylation profiles and electronic medical records, we could design a biomarker panel that allows for early and accurate noninvasive detection of CRC using stool samples.

Development of a highly sensitive chemiluminescent enzyme immunoassay for fragmented cytokeratin 18 using new antibodies.

Fragmented cytokeratin 18 (fCK18) released from epithelial cells undergoing apoptosis is widely studied in various diseases. However, fCK18 measurement is not utilized in clinical practice due to imprecise disease-state cutoff values. Therefore, we set out to generate new monoclonal antibodies (mAbs) and a recombinant fCK18 (rfCK18) calibrator in an effort to develop a highly sensitive chemiluminescent enzyme immunoassay (CLEIA). New capture mAb (K18-624) had a high binding ability compared to the current commercial antibody. New detection mAb (K18-328) recognized 323S-340G of CK18. A rfCK18 was expressed in the soluble fraction of E. coli when the N-terminal region (260 amino acid residues) of CK18 was truncated. Analysis of performance and measurement of human fCK18 were evaluated using K18-624 and K18-328 in a highly sensitive CLEIA. The coefficients of variation (CV) for within-run and between-day repeatability were below 10% and the recoveries were in the range of 15%. The detection sensitivity was 0.056 ng/mL. Serum fCK18 levels were significantly increased in non-alcoholic steatohepatitis (NASH) patients when compared to healthy individuals. Our new fCK18 mAbs showed high affinity and sensitivity. CLEIA using our new antibodies will be useful in measuring fCK18 in human blood thereby generating accurate clinical diagnoses of human liver diseases.

Diagnostic Accuracy of HPV Detection in Patients with Oropharyngeal Squamous Cell Carcinomas: A Systematic Review and Meta-Analysis.

The aim of the study was to evaluate the diagnostic accuracy of Human Papillomavirus (HPV) techniques in oropharyngeal cancer. PubMed, EMBASE, the Cochrane Library and clinicaltrials.org were systematically searched for studies reporting methods of HPV detection. Primary outcomes were sensitivity and specificity of HPV detection. In this case, 27 studies were included (n = 5488, 41.6% HPV+). In this case, 13 studies evaluated HPV detection in tumour tissue, nine studies examined HPV detection in blood samples and five studies evaluated HPV detection in oral samples. Accuracy of HPV detection in tumour tissue was high for all detection methods, with pooled sensitivity ranging from 81.1% (95% CI 71.9-87.8) to 93.1% (95% CI 87.4-96.4) and specificity ranging from 81.1% (95% CI 71.9-87.8) to 94.9% (95% CI 79.1-98.9) depending on detection methods. Overall accuracy of HPV detection in blood samples revealed a sensitivity of 81.4% (95% CI 62.9-91.9) and a specificity of 94.8% (95% CI 91.4-96.9). In oral samples pooled sensitivity and specificity were lower (77.0% (95% CI 68.8-83.6) and 74.0% (95% CI 58.0-85.4)). In conclusion, we found an overall high accuracy for HPV detection in tumour tissue regardless of the HPV detection method used. HPV detection in blood samples may provide a promising new way of HPV detection.

Assessment of MYC/PTEN Status by Gene-Protein Assay in Grade Group 2 Prostate Biopsies.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non-organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74-24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

Development and evaluation of a loop-mediated isothermal amplification assay for clinical diagnosis of respiratory human adenoviruses emergent in China.

Three human adenovirus (HAdV) genotypes, HAdV-7, HAdV-14, and HAdV-55, emerged as the most prevalent variants in China over the past decade and caused both sporadic, fatal cases and frequent, large outbreaks. Early diagnosis is essential to control infections and endemics. Here, we established a loop-mediated isothermal amplification (LAMP) assay coupled with an instrument-free nucleic acid extraction device recently developed by our group; the assay could detect all the 3 prevalent HAdV genotypes. Specificity analysis showed no cross-reactivity with other common respiratory pathogens and the analytical sensitivity was as low as 10 copies/µL. All detection steps could be completed within 1 hour. The assay's performance was evaluated using clinical samples and compared with the gold standard RT-PCR method, showing highly consistent results. The LAMP assay developed here could be readily used in basic laboratory facilities and with minimal DNA extraction equipment, and as a reliable screening test in a resource-limited setting.

Prospective high-throughput genome profiling of advanced cancers: results of the PERMED-01 clinical trial.

BACKGROUND: The benefit of precision medicine based on relatively limited gene sets and often-archived samples remains unproven. PERMED-01 (NCT02342158) was a prospective monocentric clinical trial assessing, in adults with advanced solid cancer, the feasibility and impact of extensive molecular profiling applied to newly biopsied tumor sample and based on targeted NGS (t-NGS) of the largest gene panel to date and whole-genome array-comparative genomic hybridization (aCGH) with assessment of single-gene alterations and clinically relevant genomic scores. METHODS: Eligible patients with refractory cancer had one tumor lesion accessible to biopsy. Extracted tumor DNA was profiled by t-NGS and aCGH. We assessed alterations of 802 "candidate cancer" genes and global genomic scores, such as homologous recombination deficiency (HRD) score and tumor mutational burden. The primary endpoint was the number of patients with actionable genetic alterations (AGAs). Secondary endpoints herein reported included a description of patients with AGA who received a "matched therapy" and their clinical outcome, and a comparison of AGA identification with t-NGS and aCGH versus whole-exome sequencing (WES). RESULTS: Between November 2014 and September 2019, we enrolled 550 patients heavily pretreated. An exploitable complete molecular profile was obtained in 441/550 patients (80%). At least one AGA, defined in real time by our molecular tumor board, was found in 393/550 patients (71%, two-sided 90%CI 68-75%). Only 94/550 patients (17%, 95%CI 14-21) received an "AGA-matched therapy" on progression. The most frequent AGAs leading to "matched therapy" included PIK3CA mutations, KRAS mutations/amplifications, PTEN deletions/mutations, ERBB2 amplifications/mutations, and BRCA1/2 mutations. Such "matched therapy" improved by at least 1.3-fold the progression-free survival on matched therapy (PFS2) compared to PFS on prior therapy (PFS1) in 36% of cases, representing 6% of the enrolled patients. Within patients with AGA treated on progression, the use of "matched therapy" was the sole variable associated with an improved PFS2/PFS1 ratio. Objective responses were observed in 19% of patients treated with "matched therapy," and 6-month overall survival (OS) was 62% (95%CI 52-73). In a subset of 112 metastatic breast cancers, WES did not provide benefit in term of AGA identification when compared with t-NGS/aCGH. CONCLUSIONS: Extensive molecular profiling of a newly biopsied tumor sample identified AGA in most of cases, leading to delivery of a "matched therapy" in 17% of screened patients, of which 36% derived clinical benefit. WES did not seem to improve these results. TRIAL REGISTRATION: ID-RCB identifier: 2014-A00966-41; ClinicalTrials.gov identifier: NCT02342158 .

Optimizing Nanopore sequencing-based detection of structural variants enables individualized circulating tumor DNA-based disease monitoring in cancer patients.

Here, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay. The results suggest that SV dynamics correlate with and may improve existing treatment-response biomarkers such as PSA. https://github.com/UMCUGenetics/SHARC .

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC.

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.

Could MicroRNAs Be Useful Tools to Improve the Diagnosis and Treatment of Rare Gynecological Cancers? A Brief Overview.

Gynecological cancers pose an important public health issue, with a high incidence among women of all ages. Gynecological cancers such as malignant germ-cell tumors, sex-cord-stromal tumors, uterine sarcomas and carcinosarcomas, gestational trophoblastic neoplasia, vulvar carcinoma and melanoma of the female genital tract, are defined as rare with an annual incidence of <6 per 100,000 women. Rare gynecological cancers (RGCs) are associated with poor prognosis, and given the low incidence of each entity, there is the risk of delayed diagnosis due to clinical inexperience and limited therapeutic options. There has been a growing interest in the field of microRNAs (miRNAs), a class of small non-coding RNAs of ∼22 nucleotides in length, because of their potential to regulate diverse biological processes. miRNAs usually induce mRNA degradation and translational repression by interacting with the 3' untranslated region (3'-UTR) of target mRNAs, as well as other regions and gene promoters, as well as activating translation or regulating transcription under certain conditions. Recent research has revealed the enormous promise of miRNAs for improving the diagnosis, therapy and prognosis of all major gynecological cancers. However, to date, only a few studies have been performed on RGCs. In this review, we summarize the data currently available regarding RGCs.

A coordinated approach for the assessment of molecular subgroups in pediatric ependymomas using low-cost methods.

Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (C11orf95-RELA, YAP1-MAMLD1, and YAP1-FAM118B) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of CXorf67, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with LAMA2/NELL2 markers. RELA cases and YAP1-MAMLD1 fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note, LAMA2 and NELL2 gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN. KEY MESSAGES: Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups. RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs. Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs. Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups. A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.

Beyond Standard Practice in Liquid Biopsy: Selective Venous Sampling.

Liquid biopsy is a molecular diagnostic procedure that aims to provide readily accessible genetic profiling of tumors for primary diagnosis, detection of minimal residual or metastatic disease, and therapeutic decision-making, especially for molecularly targeted treatments. Cancers release various biological markers into the circulation, although the most widely used are cell-free tumor DNA and circulating tumor cells. The paucity of biological material means that laboratory methods mainly based on genetic sequencing expose this innovative diagnostic method to a considerable incidence of false negatives. The 3 cases presented here show how the sensitivity and specificity of liquid biopsy may be improved through selective venous sampling.

ESGO/ESTRO/ESP Guidelines for the management of patients with endometrial carcinoma.

A European consensus conference on endometrial carcinoma was held in 2014 to produce multidisciplinary evidence-based guidelines on selected questions. Given the large body of literature on the management of endometrial carcinoma published since 2014, the European Society of Gynaecological Oncology (ESGO), the European SocieTy for Radiotherapy & Oncology (ESTRO) and the European Society of Pathology (ESP) jointly decided to update these evidence-based guidelines and to cover new topics in order to improve the quality of care for women with endometrial carcinoma across Europe and worldwide. ESGO/ESTRO/ESP nominated an international multidisciplinary development group consisting of practicing clinicians and researchers who have demonstrated leadership and expertise in the care and research of endometrial carcinoma (27 experts across Europe). To ensure that the guidelines are evidence-based, the literature published since 2014, identified from a systematic search was reviewed and critically appraised. In the absence of any clear scientific evidence, judgment was based on the professional experience and consensus of the development group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 191 independent international practitioners in cancer care delivery and patient representatives. The guidelines comprehensively cover endometrial carcinoma staging, definition of prognostic risk groups integrating molecular markers, pre- and intra-operative work-up, fertility preservation, management for early, advanced, metastatic, and recurrent disease and palliative treatment. Principles of radiotherapy and pathological evaluation are also defined.