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Molecular Diagnostics Literature Review

Genome-wide analysis of circulating cell-free DNA copy number detects active melanoma and predicts survival
Silva S, Danson S, Teare D, Taylor F, Bradford J, et al. Clin Chem 2018; 64(9): 1338–1346.

BACKGROUND: A substantial number of melanoma patients develop local or metastatic recurrence, and early detection of these is vital to maximize benefit from new therapies such as inhibitors of BRAF and MEK, or immune checkpoints. This study explored the use of novel DNA copy-number profiles in circulating cell-free DNA (cfDNA) as a potential biomarker of active disease and survival.

PATIENTS AND METHODS: Melanoma patients were recruited from oncology and dermatology clinics in Sheffield, UK, and cfDNA was isolated from stored blood plasma. Using low-coverage whole-genome sequencing, we created copy-number profiles from cfDNA from 83 melanoma patients, 44 of whom had active disease. We used scoring algorithms to summarize copy-number aberrations and investigated their utility in multivariable logistic and Cox regression analyses.

RESULTS: The copy-number aberration score (CNAS) was a good discriminator of active disease (odds ratio, 3.1; 95 % CI, 1.5–6.2; P=0.002), and CNAS above or below the 75th percentile remained a significant discriminator in multivariable analysis for active disease (P=0.019, with area under ROC curve of 0.90). Additionally, mortality was higher in those with CNASs above the 75th percentile than in those with lower scores (HR, 3.4; 95 % CI, 1.5–7.9; P=0.005), adjusting for stage of disease, disease status (active or resected), BRAF status, and cfDNA concentration.

CONCLUSIONS: This study demonstrates the potential of a de novo approach utilizing copy-number profiling of cfDNA as a biomarker of active disease and survival in melanoma. Longitudinal analysis of copy-number profiles as an early marker of relapsed disease is warranted.

Microbiological diagnostics of bloodstream infections in Europe – An ESGBIES survey
Idelevich EA, Seifert H, Sundqvist M, Scudeller L, Amit S, et al. Clin Microbiol Infect 2019; doi: 10.1016/j.cmi.2019.03.024 [Epub ahead of print].

OBJECTIVES: High-quality diagnosis of bloodstream infections (BSIs) is important for successful patient management. As knowledge on current practices of microbiological BSI diagnostics is limited, this project aimed to assess its current state in European microbiological laboratories.

METHODS: We performed an online questionnaire-based cross-sectional survey comprising 34 questions on practices of microbiological BSI diagnostics. The ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES) was the primary platform to engage national coordinators who recruited laboratories within their countries.

RESULTS: Responses were received from 209 laboratories in 25 European countries. While 32.5 % (68/209) of laboratories only used the classical processing of positive blood cultures (BCs), two-thirds applied rapid technologies. Of laboratories that provided data for respective question, 42.2 % (78/185) were able to start incubating blood cultures in automated BC incubators around-the-clock, and only 13 % (25/192) had established a 24-hour service to start immediate processing of positive BCs. Only 4.7 % (9/190) of laboratories validated and transmitted the results of identification and antimicrobial susceptibility testing (AST) of BC pathogens to clinicians 24 hours/day. MALDI-TOF MS from shortly incubated sub-cultures on solid media was the most commonly used approach to rapid pathogen identification from positive BCs, and direct disk diffusion was the most common rapid AST method from positive BCs.

CONCLUSIONS: Laboratories have started to implement novel technologies for rapid identification and AST for positive BCs. However, progress is severely compromised by limited operating hours such that current practice of BC diagnostics in Europe complies only partly with the requirements for optimal BSI management.

An integrated next-generation sequencing system for analyzing DNA mutations, gene fusions, and RNA expression in lung cancer
Haynes BC, Blidner RA, Cardwell RD, Zeigler R, Gokul S, et al. Transl Oncol 2019; 12(6): 836–845.

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non-small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97 % agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Molecular diagnosis of asparagine synthetase (ASNS) deficiency in two Indian families and literature review of 29 ASNS deficient cases
Devi ARR, Naushad SM. Gene 2019; doi: 10.1016/j.gene.2019.04.024 [Epub ahead of print].

In the current study, we report three cases of asparagine synthetase (ASNS) deficiency from two consanguineous families. Family 1 had two early neonatal deaths due to a novel mutation in the ASNS gene c.788C > T (p.S263F) and both the children presented with microcephaly and one of them had severe intracranial hemorrhage. The proband from the second family was homozygous for c.146G > A (p.R49Q) and manifested myoclonic seizures, developmental delay, coarse hair and diffuse cortical atrophy. Molecular docking studies of both the mutations revealed alteration in the ligand binding site. To date, 26 mutations were reported in ASNS gene in 29 affected children indicating high degree of genetic heterogeneity and high mortality. Although asparagine depletion is not of diagnostic utility, multiple linear regression model suggested that asparagine levels vary to the extent of 20.6 % based on glutamine and aspartate levels and ASNS deficiency results in depletion of arginine synthesis. ASNS deficiency should be suspected in any neonate with microcephaly and epileptic encephalopathy.