Role Of Next-generation Sequencing Research Articles

Role of next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder.

Next generation sequencing based diagnosis has emerged as a promising tool for evaluating critically ill neonates and children. However, there is limited data on its utility in developing countries. We assessed its diagnostic rate and clinical impact on management of pediatric patients with a suspected genetic disorder requiring critical care. The study was conducted at a single tertiary hospital in Northern India. We analyzed 70 children with an illness requiring intensive care and obtained a precise molecular diagnosis in 32 of 70 probands (45.3%) using diverse sequencing techniques such as clinical exome, whole exome, and whole genome. A significant change in clinical outcome was observed in 13 of 32 (40.6%) diagnosed probands with a change in medication in 11 subjects and redirection to palliative care in two subjects. Additional benefits included specific dietary management (three cases), avoidance of a major procedure (one case) and better reproductive counseling. Dramatic therapeutic responses were observed in three cases with SCN1A, SCN2A and KCNQ2-related epileptic encephalopathy. A delayed turn-around for sequencing results was perceived as a major limiting factor in the study, as rapid and ultra-rapid sequencing was not available. Achieving a precise molecular diagnosis has great utility in managing critically ill patients with suspected genetic disorders in developing countries.

Reclassification of a spindle cell sarcoma after identification of a TFG-ROS1 fusion: A case demonstrating the clinical benefit of next-generation sequencing in sarcoma.

Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next-generation sequencing (NGS) for classification of unselected sarcomas remains controversial. We report a case of a metastatic sarcoma in a 34-year-old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG-ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence insitu hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG-ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK-negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment.

The Role of Next-Generation Sequencing in the Management of Patients with Suspected Non-Ischemic Cardiomyopathy after Syncope or Termination of Sudden Arrhythmic Death.

Cardiac arrhythmias and sudden death are frequent in patients with non-ischemic cardiomyopathy and can precede heart failure or additional symptoms where malignant cardiac arrhythmias are mostly the consequence of advanced cardiomyopathy and heart failure. Finding these subgroups and making an early diagnosis could be lifesaving. In our retrospective study, we are presenting arrhythmic types of frequent cardiomyopathies where an arrhythmogenic substrate is less well defined, as in ischemic or structural heart disease. In the period of 2 years, next-generation sequencing (NGS) tests along with standard clinical tests were performed in 208 patients (67 women and 141 men; mean age, 51.2 ± 19.4 years) without ischemic or an overt structural heart disease after syncope or aborted sudden cardiac death. Genetic variants were detected in 34.4% of the study population, with a significant proportion of pathogenic variants (P) (14.4%) and variants of unknown significance (VUS) (20%). Regardless of genotype, all patients were stratified according to clinical guidelines for aggressive treatment of sudden cardiac death with an implantable cardioverter defibrillator (ICD). The P variant identified by NGS serves for an accurate diagnosis and, thus, better prevention and specific treatment of patients and their relatives. Results in our study suggest that targeted sequencing of genes associated with cardiovascular disease is an important addendum for final diagnosis, allowing the identification of a molecular genetic cause in a vast proportion of patients for a definitive diagnosis and a more specific way of treatment. VUS in this target population poses a high risk and should be considered possibly pathogenic in reanalysis.

Clinical Outcomes of Crizotinib Readministration in Patients with Nonsmall Cell Lung Cancer with Anaplastic Lymphoma Kinase Rearrangement: Case Report and Review of Literature

AbstractRecent studies have demonstrated promising outcomes of the first-line anaplastic lymphoma kinase-tyrosine kinase inhibitor (ALK-TKI) “crizotinib” in patients with locally advanced and metastatic lung cancers with high expression of the fusion protein “EML4-ALK.” High drug resistance, however, restricts the therapeutic advantages of ALK-TKIs in patients with nonsmall cell lung cancer (NSCLC). The contemporary literature documents limited treatment approaches for patients with NSCLC relapse or nonresponsiveness to second-/third-generation ALK-TKIs. We hereby provide a descriptive analysis of five NSCLC cases treated with crizotinib, ceritinib, and alectinib for a median duration of 54 months. The outcomes indicate a profound therapeutic response in patients receiving 4th and subsequent line of treatment with crizotinib. The crizotinib retreatment actively reduced patient resistance to the ALK-TKIs by reversing the mesenchymal epithelial transition amplification. The results from this case series also emphasize the possible role of next-generation sequencing in determining therapeutic resistance and transforming the treatment paradigm for NSCLC. Partial response was observed in the patients after 6 months of crizotinib readministration. This is possibly the first case series reporting crizotinib rechallenge in patients of ALK positive NSCLC who failed on subsequent ALK-TKIs and multiple lines of chemotherapies.

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine.

Next-generation sequencing (NGS) has modernized the field of tuberculosis (TB) research by enabling high-throughput sequencing of the entire genome of Mycobacterium tuberculosis (MTB), which is the causative agent of TB. NGS has provided insights into the genetic diversity of MTB, which are crucial for understanding the evolution and transmission of the disease, and it has facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. However, the high cost and the technical complexities of NGS currently limit its widespread use in clinical settings. International recommendations are thus necessary to facilitate the interpretation of polymorphisms, and an experimental approach is still necessary to correlate them to phenotypic data. This review aims to present a comparative, step-by-step, and up-to-date review of the techniques available for the implementation of this approach in routine laboratory workflow. Ongoing research on NGS for TB holds promise for improving our understanding of the disease and for developing more efficacious treatments.

THE ROLE OF NEXT-GENERATION SEQUENCING IN LUNG CANCER DIAGNOSIS

Among all malignant neoplasms, lung cancer is the cause of death in approximately every fifth patient. Next-generation sequencing can solve the issue of not only diagnosis but also the further treatment of lung cancer. Aim. The work aims to search, process, generalize and bibliometrically analyze the scientific literature to study the main trends in next-generation sequencing in diagnosing non-small cell lung cancer. Materials and methods. One thousand nine hundred thirty-one literature sources, including information about next-generation sequencing, were published between 2010 and 2023. The data search was carried out in electronic databases Scopus, PubMed, Web of Science, and Google Scholar using keywords: "lung cancer," "next-generation sequencing," "targeted therapy," "genetic mutations," "circulating tumor DNA," and "liquid biopsy." The authors used the bibliometric tools of the Scopus and SciVal databases to analyze the year, source, type of study, subject area, and country of publication. Results and discussion. Next-generation sequencing is gradually becoming the new diagnostic standard. This technology allows to detect biological markers with high accuracy and specificity. Many studies have confirmed the effectiveness of next-generation sequencing for diagnosing lung cancer, assessing response to treatment and sensitivity to drug therapy, and predicting the prognosis of the disease. Lung cancer is one of the most common tumors with a high mutational load. Unique diagnostic panels allow for a short period to examine tumor tissue for a wide range of biological markers. The article aims to investigate the main areas of application of next-generation sequencing in patients with lung cancer and current clinical trials in this field. The bibliometric analysis of the scientific literature consisted of the study of publication activity from 2010 to 2023, the geography of publications, and the identification of scientific journals where the articles about the role of next-generation sequencing were publicized. Conclusions. Next-generation sequencing is widely used in medicine. This method can become one of the leading methods for diagnosing lung cancer because it can accurately identify specific biological markers. Tumor tissue embedded in paraffin blocks and various biological fluids can be used for diagnosis. Next-generation sequencing is effective even in cases where the amount of tumor tissue is limited and other methods cannot identify it. The advantages of the method are confirmed in clinical trials and described in many scientific publications of the leading countries of the world. As a result, next-generation sequencing appears to be an effective method for diagnosing lung cancer and selecting the most appropriate regimen of targeted or immunotherapy. Disadvantages of the method include the high cost in developing countries and the need for appropriate software.

The role of next-generation sequencing in the examination of signaling genes in Brca1/2-negative breast cancer cases.

Breast cancer is the most prevalent malignancy in women worldwide. Although pathogenic variants in the BRCA1/2 genes are responsible for the majority of hereditary breast cancer cases, a substantial proportion of patients are negative for pathogenic variations in these genes. In cancers, the signal transduction pathways of the cell are usually affected first. Therefore, this study aimed to detect and classified genetic variations in non-BRCA signaling genes and investigate the underlying genetic causes of susceptibility to breast cancer. Ninety-six patients without pathogenic variants in the BRCA1/2 genes who met the inclusion criteria were enrolled in the study, and 34 genes were analyzed using next-generation sequencing (NGS) for genetic analysis. Based on the ClinVar database or American College of Medical Genetics criteria, a total of 55 variants of 16 genes were detected in 43 (44.8%) of the 96 patients included in the study. The pathogenic variants were found in the TP53, CHEK2, and RET genes, whereas the likely pathogenic variants were found in the FGFR1, FGFR3, EGFR, and NOTCH1 genes. The examination of signaling genes in patients who met the established criteria for hereditary breast cancer but were negative for BRCA1/2 pathogenic variants provided additional information for approximately 8% of the families. The results of the present study suggest that NGS is a powerful tool for investigating the underlying genetic causes of occurrence and progression of breast cancer.

Diagnosing Czech Patients with Inherited Platelet Disorders.

A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.

The role of next-generation sequencing in acute myeloid leukemia.

The development of high-throughput techniques like next-generation sequencing (NGS) has unraveled the genetic profile of cancer. In this review, we discuss the role of NGS on the diagnostic, risk stratification, and follow-up of patients with acute myeloid leukemia (AML). NGS has become an essential tool in clinical practice for AML management. Therefore, efforts are being made to improve its applications, automation, and turnaround time. Other high-throughput techniques, such as whole genome sequencing or RNA-sequencing, can be also used to this end. However, not all institutions may be able to implement these approaches. NGS is being investigated for measurable residual disease (MRD) assessment, especially with the development of error-correction NGS. New data analysis approaches like machine learning are being investigated in order to integrate genomic and clinical data and develop comprehensive classifications and risk scores. NGS has proven to be a useful approach for the analysis of genomic alterations in patients with AML, which aids patient management. Current research is being directed at reducing turnaround time and simplifying processes so that these techniques can be universally integrated into clinical practice.

Clinicopathologic Characteristics of Grade 2/3 Meningiomas: A Perspective on the Role of Next-Generation Sequencing.

BackgroundGrade 2/3 meningiomas have locally aggressive behaviors often requiring additional treatment plans after surgical resection. Herein, we explored the clinical significance of next-generation sequencing (NGS) in characterizing the molecular profiles of high-grade meningiomas.MethodsPatients with intracranial meningioma who underwent surgical resection in a single institution were retrospectively reviewed. Clinicopathologic relevance was evaluated using recurrence-free survival (RFS) as an outcome measure. NGS for the targeted gene regions was performed in 40 participants.ResultsAmong the 713 individuals in the study population, 143 cases (20.1%) were identified as having grade 2 or 3 meningiomas with a significantly lower female predominance. While the difference in RFS between grade 2 and 3 meningiomas was insignificant, a few conventional grade 2 cases, but with TERT promoter hotspot mutation, were highly progressive and refractory to the treatment. From the NGS study, recurrent mutations in TRAF and AKT1 were identified with a higher prevalence (17.5% and 12.5%, respectively) compared with grade 2/3 meningiomas reported in previous literature. However, their relations to other histopathologic properties or clinical factors were rarely observed.ConclusionsGrade 2/3 meningiomas show a broad spectrum of molecular profiles, as they have heterogeneous histologic characteristics.

Therapeutic Advances and Challenges in the Management of HER2-Positive Gastroesophageal Cancers.

Gastroesophageal cancer is one of the most common cancers in the world, with a high rate of mortality. While there has been significant progress over the past decade, particularly with the addition of anti-HER2 therapies to platinum-based chemotherapy agents in the advanced setting, the prognosis remains poor and the treatment options for this disease entity remain limited. In this review, we discuss the current therapeutic landscape for HER2-positive gastroesphageal cancer and the seminal clinical trials that have shaped our approach to this disease entity. In addition, we highlight some of the challenges to the understanding and management of this disease, specifically discussing the breadth of molecular diversity and intratumoral heterogeneity of HER2 expression that impact the clinical efficacy and prognosis. Furthermore, we discuss the potential role of next-generation sequencing (NGS) and circulating-tumor DNA (ctDNA) as complementary tools to immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) to guiding clinical decision making. Finally, we highlight promising clinical trials of new treatment regimens that will likely reshape the therapeutic approach to this disease entity.

Mini-Review: Pancreatic Cancer Presenting as a Bowel Obstruction and the Role of Next-Generation Sequencing

Pancreatic adenocarcinoma has increasingly become one of the leading causes of death in western countries. However, presentation with colonic metastases is far less frequently reported in the literature and may be misdiagnosed as colonic adenocarcinoma. This is a case of a female patient with metastatic pancreatic adenocarcinoma that presented with a sigmoid obstruction.

Novel biomarkers and the future of targeted therapies in cholangiocarcinoma: a narrative review.

Cholangiocarcinoma is a highly aggressive and heterogenous group of biliary malignancies arising from any site in the biliary tree, comprising 15% of all primary liver cancers. The nature of the disease and nonspecific presentation leads to late diagnosis and ultimately poor outcomes for patients. Combination gemcitabine and cisplatin has been the standard of care for cholangiocarcinoma (CCA) since 2010, with a median overall survival of 11.7 months. The five-year survival for CCA remains 5-10%, revealing a clear need for improved treatment options. This targeted review highlights the role of next generation sequencing in CCA and the clinically relevant tumor biomarkers that have become the focus of therapeutic development. These tumor biomarkers or actionable mutations hold the potential to enable earlier diagnosis, provide prognostic information, and guide treatment decisions for patients with CCA. Specifically, the FGFR2 fusion and IDH1 mutation have shown considerable promise in development of targeted therapies. Clinical trials with inhibitors targeting FGFR2 fusion and IDH1 mutation have created expectations that these drugs will soon enter clinical practice. Other biomarkers including KRAS and B-raf protooncogenes, Her2/neu genes, and BRCA1 and 2 tumor-suppressor genes have also been touted as potential targets for future therapies, with early data showing promise for new drug development. The discovery of these actionable mutations and identification of targeted therapies have challenged the notion of a "one-size fits all" for treatment of CCA, and generated optimism that these novel treatments will soon be available for patients with CCA.

The role of next-generation sequencing in bile of patients with biliary stenosis secondary to pancreatic ductal adenocarcinoma

The role of next-generation sequencing in bile of patients with biliary stenosis secondary to pancreatic ductal adenocarcinoma

Next Generation Sequencing and Molecular Biomarkers in Ovarian Cancer-An Opportunity for Targeted Therapy.

Ovarian cancer is the deadliest of all gynecologic malignancies claiming the lives of nearly 14,000 women in the United States annually. Despite therapeutic advances, the ovarian cancer mortality rate has remained stagnant since the 1980’s. The molecular heterogeneity of ovarian cancers suggest they may be more effectively treated via precision medicine. Current guidelines recommend germline and somatic testing for all new epithelial ovarian cancer diagnoses to assist providers in identifying candidates for targeted therapies. Next generation sequencing (NGS) identifies targetable, driver, and novel mutations used to guide treatment decisions. Performing NGS is standard of care in many other malignancies, but for ovarian cancer the use of NGS in daily practice is still emerging. This review discusses the targetable genetic mutations and role of NGS and molecular biomarker testing in the treatment of ovarian cancer.

Next-generation Sequencing: For the Present Generation Oncologist

Previously, doctors had to treat this deadly disease based on immunohistochemistry, polymerase chain reaction and fluorescence in situ hybridisation-based diagnostics tests information which used to provide limited information. There was always a requirement of a single test/assay which could give all these information in a single assay and without much loss of precious sample. Comprehensive next-generation sequencing (NGS)-based assays are gaining much importance in treatment decision among clinicians. These present technologies using DNA, RNA, and methylation sequencing have brought a lot of changes in cancer therapeutics. Oncogenic drivers are genomic alteration lead to malignant transformation and progression of cancer. The interpretation of the results of NGS is also very challenging as the results obtained are ‘variants’ are of unknown prognostic significance. Apart from these, NGS helps us in documenting various other genomic signatures such as tumour mutation burden and microsatellite instability. There are now multiple gene panels that are recommended by major international societies such as NCCN, for the treatment of various malignancies. In fact, NGS forms the most important pillar of precision oncology and has brought a paradigm shift in the way major cancers are treated. It is still an evolving field and many times the interpretation of the results of NGS reports is very difficult. NGS-based precision medicine treatment offers true value addition in clinical practice has a positive impact on patient lives in cases of refractory cancer, the role of NGS is the only source of light that helps us in navigating amidst the darkness and hopelessness of a refractory malignancy. In fact, presently not only in refractory malignancies but the role of NGS has come in the management of cancer patients in the first line as it gives us a more comprehensive understanding of the disease. This review article has been written with an idea to make a general practitioner aware of this novel technique, the advantages as well as the pitfalls.

EP465: Role of next-generation sequencing in the evaluation of families with non-immune fetal hydrops

Fetal hydrops, which refers to the pathological accumulation of fluid in the fetal soft tissues and serous cavities, is an important cause of prenatal and perinatal morbidity and mortality. Non-immune fetal hydrops (NIFH) is an etiologically heterogeneous condition, which can occur due to genetic as well as non-genetic causes. Genetic causes for NIFH include numerical and structural chromosomal anomalies, and many different monogenic conditions including inborn errors of metabolism (IEMs), lethal skeletal dysplasias, hematological disorders, multiple malformation syndromes, etc.

The role of next-generation sequencing in detecting gene fusions with known and unknown partners: a single-center experience with methodologies’ integration

Next-generation sequencing (NGS) is becoming a new gold standard for determining molecular predictive biomarkers. This study aimed to evaluate the reliability of NGS in detecting gene fusions, focusing on comparing gene fusions with known and unknown partners. We collected all gene fusions from a consecutive case series using an amplicon-based DNA/RNA NGS platform and subdivided them into two groups: gene fusions with known partners and gene fusions with unknown partners. Gene fusions involving ALK, ROS1 and RET were also examined by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH). Overall, 1174 malignancies underwent NGS analysis. NGS detected gene fusions in 67 cases (5.7%), further subdivided into 43 (64.2%) with known partners and 24 (35.8%) with unknown partners. Gene fusions were predominantly found in non-small cell lung carcinomas (52/67, 77.6%). Gene fusions with known partners frequently involved ALK (20/43, 46.5%) and MET (9/43, 20.9%), while gene fusions with unknown partners mostly involved RET (18/24, 75.0%). FISH/IHC confirmed rearrangement status in most (89.3%) of the gene fusions with known partners, but in only one (4.8%) of the gene fusions with unknown partners, with a significant difference (p<0.001). In 17 patients undergoing targeted therapy, the log-rank test revealed that the overall survival was higher in the known partner group than in the unknown partner group (p=0.002). NGS is a reliable method for detecting gene fusions with known partners, but it is less accurate in identifying gene fusions with unknown partners, for which further analyses (such as FISH) are required.

Somatic Mutation Profiles Revealed by Next Generation Sequencing (NGS) in 39 Chinese Hepatocellular Carcinoma Patients.

The features and significance of somatic mutation profiles in hepatocellular carcinoma (HCC) have not been completely elucidated to date. In this study, 39 tumor specimens from HCC patients were collected for gene variation analysis by next-generation sequencing (NGS), and a correlation analysis between mutated genes and clinical characteristics was also conducted. The results were compared with genome data from cBioPortal database. Our study found that T > G/A > C transversions (Tv) and C > T/G > A transitions (Ti) were dominant. The sequence variations of TP53, MUC16, MUC12, MUC4 and others, and the copy number variations (CNVs) of FGF3, TERT, and SOX2 were found to be more frequent in our cohort than in cBioPortal datasets, and they were highly enriched in pathways in cancer and participated in complex biological regulatory processes. The TP53 mutation was the key mutation (76.9%, 30/39), and the most common amino acid alteration and mutation types were p.R249S (23.5%) and missense mutation (82.3%) in the TP53 variation. Furthermore, TP53 had more co-mutations with MUC17, NBPF10, and AHNAK2. However, there were no significant differences in clinical characteristics between HCC patients with mutant TP53 and wild-type TP53, and the overall survival rate between treatment via precision medication guided by NGS and that via empirical medication (logrank p = 0.181). Therefore, the role of NGS in the guidance of personalized targeted therapy, solely based on NGS, may be limited. Multi-center, large sample, prospective studies are needed to further verify these results.

Role of next-generation sequencing in diagnosing, tracking and vaccine development of severe acute respiratory syndrome coronavirus 2

An objective method of detecting infections without the requirement for clinical hypotheses is provided using next-generation sequencing (NGS) technology for the diagnosis of infectious disorders. In order to inform nations and the general public about any potential changes that may be required to respond to the variant and stop its spread, the World Health Organization and its international networks of experts have been continuously monitoring changes to the virus based on NGS data throughout the current pandemic. When tracking ongoing outbreaks, monitoring for novel pathogens or spotting potentially harmful variations of well-known diseases, NGS offers substantial advantages. Due to the technology's quick creation of high-resolution sequence data, researchers and research teams can access it easily and share it with one another. Numerous candidate vaccines have been created on several platforms quickly. Many of them have worked in crisis circumstances in numerous nations worldwide. The breakthrough infections could only be tracked by the use of NGS technology. In this review, we discussed in silico analysis using current bioinformatics approaches, and sequencing reveals unique emerging severe acute respiratory syndrome coronavirus 2 variations which have the potential to cause novel illnesses in the future.