Molecular Diagnosis & Therapy

First Responder to Genomic Information: A Guide for Primary Care Providers Abstract With rapid advances in genetics and genomics, the commercialization and access to new applications has become more widespread and omnipresent throughout biomedical research. Thus, increasingly, more patients will have personal genomic information they may share with primary care providers (PCPs) to better understand the clinical significance of the data. To be able to respond to patient inquiries about genomic data, variant interpretation, disease risk, and other issues, PCPs will need to be able to increase or refresh their awareness about genetics and genomics, and identify reliable resources to use or refer patients. While provider educational efforts have increased, with the rapid advances in the field, ongoing efforts will be needed to prepare PCPs to manage patient needs, integrate results into care, and refer as indicated.

A Novel Modified System of Simplified Chinese Criteria for Familial Hypercholesterolemia (SCCFH) Abstract Background and Objective The most significant clinical implication of familial hypercholesterolemia (FH) is early-onset coronary artery disease (CAD), highlighting the importance of a definitive diagnosis being available. Unfortunately, the existing algorithms are complex and it is often difficult to obtain information on the patient's family history. Hence, we aimed to establish a novel system of Simplified Chinese Criteria for FH (SCCFH). Methods We recruited 12,921 participants undergoing routine blood collection from November 2011 to June 2018. Clinical characteristics, laboratory examination, and genetic testing were obtained. FH was diagnosed based on the Simon Broome (SB) criteria, Dutch Lipid Clinic Network (DLCN) criteria, and SCCFH. The sensitivity, specificity, and agreement of SCCFH to these existing criteria were investigated. Results Of 12,921 participants reviewed, the prevalence of definite FH was 223 (1.73%), 202 (1.56%), and 205 (1.59%) based on the DLCN, SB, and SCCFH approaches, respectively. Compared with the DLCN and SB criteria, the SCCFH showed high sensitivity (91.9% and 100%), high specificity (100% and 99.9%), and good agreement (κ = 0.958 and 0.993). Similar results were found in several relevant clinical subgroups. Conclusions The SCCFH system is comparable to the existing criteria with high levels of sensitivity and specificity, and is easier to use clinically. Further larger prospective studies are needed to evaluate the feasibility and reliability of this system.

Overexpression of S100A4 Predicts Migration, Invasion, and Poor Prognosis of Hypopharyngeal Squamous Cell Carcinoma Abstract Introduction Hypopharyngeal squamous cell carcinoma (HSCC) is among the most lethal tumors encountered in the head and neck and frequently involves regional metastasis. However, the mechanism underlying the aggressiveness of HSCC remains elusive. S100A4 is a well-established metastasis-promoting regulator in a variety of malignancies, but its role in HSCC has not yet been identified. Objectives Our objectives were to explore the expression levels of S100A4 in HSCC tumors and its association with clinicopathological parameters and the clinical prognosis of HSCC and to confirm its role in the metastatic process of the HSCC FaDu cell line in vitro. Methods We assessed the expression levels of S100A4 with immunohistochemistry (IHC) in HSCC tumors (n = 71) and adjacent normal tissues (n = 44). In vitro experiments were performed to explore the impact of S100A4 knockdown on biological phenotypes of human HSCC FaDu cell line, including migration, invasion, proliferation, apoptosis, and cell cycle. Results The expression of S100A4 was elevated in HSCC tumors compared with adjacent normal tissues and positively correlated with cervical lymph node metastasis in this HSCC patient cohort. In vitro experiments showed that S100A4 knockdown significantly impaired migration and invasion and increased the proportion of cells in G0/G1 phase with no change in proliferation or apoptosis in FaDu cells. Additionally, nuclear S100A4 expression proved to be an independent prognostic indicator in patients with HSCC. Conclusion This study demonstrated for the first time that S100A4 expression is upregulated in HSCC tumors and that this upregulation is positively correlated with cervical lymph node metastasis of this malignancy. The metastasis-promoting role of S100A4 was further validated in the HSCC FaDu cell line, indicating that S100A4 is a potential therapeutic target for HSCC. Furthermore, this study suggests that nuclear S100A4 expression could be considered a prognostic biomarker for HSCC.

Modeling the Outcome of Systematic TPMT Genotyping or Phenotyping Before Azathioprine Prescription: A Cost-Effectiveness Analysis Abstract Background Thiopurine S-methyltransferase (TPMT) testing, either by genotyping or phenotyping, can reduce the incidence of adverse severe myelotoxicity episodes induced by azathioprine. The comparative cost-effectiveness of TPMT genotyping and phenotyping are not known. Objective Our aim was to assess the cost-effectiveness of phenotyping-based dosing of TPMT activity, genotyping-based screening and no screening (reference) for patients treated with azathioprine. Methods A decision tree was built to compare the conventional weight-based dosing strategy with phenotyping and with genotyping using a micro-simulation model of patients with inflammatory bowel disease from the perspective of the French health care system. The time horizon was set up as 1 year. Only direct medical costs were used. Data used were obtained from previous reports, except for screening test and admission costs, which were from real cases. The main outcome was the cost-effectiveness ratios, with an effectiveness criterion of one averted severe myelotoxicity episode. Results The total expected cost of the no screening strategy was €409/patient, the total expected cost of the phenotyping strategy was €427/patient, and the total expected cost of the genotyping strategy was €476/patient. The incremental cost-effectiveness ratio was €2602/severe myelotoxicity averted in using the phenotyping strategy, and €11,244/severe myelotoxicity averted in the genotyping strategy compared to the no screening strategy. At prevalence rates of severe myelotoxicity > 1%, phenotyping dominated genotyping and conventional strategies. Conclusion The phenotype-based strategy to screen for TPMT deficiency dominates (cheaper and more effective) the genotype-based screening strategy in France. Phenotype-based screening dominates no screening in populations with a prevalence of severe myelosuppression due to azathioprine of > 1%.

Aptamers as Therapeutic Agents: Has the Initial Euphoria Subsided? Abstract Aptamers are synthetic DNA or RNA oligonucleotide ligands with great potential for therapeutic applications. A vast number of disease-related targets have been used to identify agonistic, antagonistic, or inhibitory aptamers, or aptamer-based targeting ligands. However, only a few aptamers have reached late-stage clinical trials so far and the commercial infrastructure is still far behind that of other therapeutic agents such as monoclonal antibodies. The desirable properties of aptamers such as selectivity, chemical flexibility, or cost-efficiency are faced by challenges, including a short half-life in vivo, immunogenicity, and entrapment in cellular organelles. Aptamer research is still in an early stage, and a deeper understanding of their structure, target interactions, and pharmacokinetics is necessary to catch up to the clinical market. In this review, we will discuss the benefits and limitations in the development of therapeutic aptamers, as well as the advances and future directions of aptamer research. The progress towards effective therapies seems to be slow, but it has not stopped and the best is yet to come.

Clinicopathological Features of ALK Expression in 9889 Cases of Non-small-Cell Lung Cancer and Genomic Rearrangements Identified by Capture-Based Next-Generation Sequencing: A Chinese Retrospective Analysis Abstract Background The clinicopathological features and genomic rearrangements of anaplastic lymphoma kinase (ALK) fusion cases have not been fully identified. Objective Our objective was to explore the status of ALK in non-small-cell lung cancer (NSCLC) specimens, to explore the relationships between ALK status and clinicopathological features and to identify genomic rearrangements via capture-based next-generation sequencing (NGS). Methods We tested 9889 NSCLC specimens for ALK status using the Ventana anti-ALK (D5F3) antibody. Clinicopathological features were analyzed and genomic rearrangements identified using capture-based NGS in 76 ALK-positive cases. Results In total, 485 specimens (4.90%) tested positive for ALK. The positivity rate was higher for adenocarcinoma samples than for non-adenocarcinoma samples (6.03 vs. 1.47%; p < 0.001) and for biopsies/cell blocks than for surgical specimens (7.00 vs. 4.16%; p < 0.001). Patient age, patient sex, specimen type, specimen histotype, and patient smoking history were all significantly correlated with ALK status. Genomic rearrangements were detected in 98.68% (75/76) of the ALK-positive samples; 89.33% (67/75) carried the canonical EML4-ALK, and the remaining samples carried only noncanonical ALK rearrangements. Four of these noncanonical ALK fusion samples were identified as carrying EML4-ALK transcripts at the RNA level. A novel fusion variant, SRD5A2-ALK, was revealed. Conclusions Younger patients with NSCLC, especially those aged < 30 years, were more likely to test positive for ALK. Positive ALK test results were more common in patients with invasive mucinous adenocarcinoma and solid-predominant invasive adenocarcinoma than in patients with other histotypes. Samples that carried only noncanonical ALK rearrangements may also have carried the canonical EML4-ALK, which was not detected by capture-based NGS. EML4-ALK transcripts might result from rare splicing mechanisms without genomic rearrangements.

Advances in Detection of Kidney Transplant Injury Abstract Early detection of graft injury after kidney transplantation is key to maintaining long-term good graft function. Graft injury could be due to a multitude of factors including ischaemia reperfusion injury, cell or antibody-mediated rejection, progressive interstitial fibrosis and tubular atrophy, infections and toxicity from the immunosuppressive drugs themselves. The current gold standard for assessing renal graft dysfunction is renal biopsy. However, biopsy is usually late when triggered by a change in serum creatinine and of limited utility in diagnosis of early injury when histological changes are equivocal. Therefore, there is a need for timely, objective and non-invasive diagnostic techniques with good early predictive value to determine graft injury and provide precision in titrating immunosuppression. We review potential novel plasma and urine biomarkers that offer sensitive new strategies for early detection and provide major insights into mechanisms of graft injury. This is a rapidly expanding field, but it is likely that a combination of biomarkers will be required to provide adequate sensitivity and specificity for detecting graft injury.

Clinicopathological Significance of Overall Frequency of Allelic Loss (OFAL) in Lesions Derived from Thyroid Follicular Cell Abstract Background Loss of heterozygosity (LOH) and microsatellite instability (MSI) are frequent molecular events in thyroid tumor etiopathogenesis occurring in several chromosomal critical areas, including 3p12–25.3, 7q21–31, 10q22–24, and 15q11–13, with loci of tumor suppressor genes. Objective We evaluated the usefulness of LOH/MSI as a diagnostic/prognostic biomarker in lesions derived from thyroid follicular cells: follicular thyroid carcinoma (FTC); follicular adenoma (FA), papillary thyroid carcinoma (PTC), and nodular goiter (NG). Methods We performed allelotyping (GeneMapper Software v. 4.0.) of ten microsatellite markers linked to the 1p31.2, 3p21.3, 3p24.2, 9p21.3, 11p15.5, and 16q22.1 region on DNA from 93 primary thyroid lesions then evaluated the LOH/MSI frequency and overall frequency of allelic loss (OFAL). Results We found regions with significantly increased frequency of LOH/MSI for specific histotypes: the 3p24.2 region for FA and 1p31.2 for FTC. LOH/MSI in 3p21.3 was significantly elevated in PTC and FTC. LOH/MSI in 3p21.3 was increased for small size tumors (T1a + T1b), tumors with no regional lymph node involvement (N0 + Nx), American Joint Committee on Cancer (AJCC) stage I tumors, and tumor diameter (Td) < 10 mm; in 1p31.2 for T2–3, N1, stage II–IV, and Td 10–30 mm; in 11p15.5 for T2–3, N1, stage II–IV, and Td > 30 mm. OFAL values were significantly higher in younger patients (< 40 years), in men, in those with T2–3 stage tumors, in those with increased Td, and in FA and FTC compared with NG and PTC. Conclusions We confirmed the occurrence of LOH/MSI in 3p21.3 at an early stage of tumorigenesis and mapped 1p31.2 and 11p15.5 as characteristic for advanced-stage tumors. The results of our study may enable consideration of OFAL, defined as LOH/MSI coincidence in various chromosomal regions, as a tumor progression marker. OFAL values were significantly higher in follicular neoplasms (FA and FTC) than in PTC or NG; hence, increased OFAL values can be regarded as a characteristic feature of the follicular phenotype.

Vortex- and Centrifugation-Free Extraction of HIV-1 RNA Abstract Background and Objective HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments. Methods A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil–water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples. Results Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%. Conclusions From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

Mutational Landscape of Resistance to Thyroid Hormone Beta (RTHβ) Abstract Resistance to thyroid hormone beta (RTHβ) is a syndrome characterized by reduced responsiveness of peripheral tissues to thyroid hormone (TH). In most cases, the disorder is associated with germline pathogenic variants in the thyroid hormone receptor beta (THRB) gene. This paper summarizes the clinical and biochemical presentation of the disease, providing a comprehensive overview on molecular genetic features. Particular care is given in reporting all identified THRB variants with an assessed or unknown clinical significance. Our aim is to offer a useful tool for clinical and genetic specialists in order to ease clinical diagnosis and genetic counseling.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com