The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

UNASSIGNED: Circulating tumor DNA (ctDNA) is released by many tumors into the plasma. Its analysis has minimal procedural risk and, in many cancers, has the potential for clinical applications. In retinoblastoma, the clinical correlations of ctDNA in eyes treated without enucleation have not been studied. This purpose of this study was to determine how the ctDNA RB1 variant allele frequency (VAF) changes in patients with unilateral retinoblastoma after intra-arterial chemotherapy (IAC) treatment. Variant allele frequency is a proxy for tumor fraction.
UNASSIGNED: Case series from a single tertiary cancer referral center.
UNASSIGNED: Five patients with retinoblastoma with at least 1 measurable ctDNA plasma specimen both at the time of active intraocular retinoblastoma before IAC and after at least 1 IAC cycle.
UNASSIGNED: Circulating tumor DNA RB1 was detected and VAF was measured before and after IAC treatment. Clinical correlations were made using clinical examination, fundus photography, ultrasound, and OCT.
UNASSIGNED: Comparison of ctDNA RB1 VAF before and after IAC treatment for retinoblastoma and concordance of ctDNA RB1 detectability with activity of intraocular disease.
UNASSIGNED: Twenty-three ctDNA specimens were included from 5 patients. The 5 baseline RB1 VAFs ranged from 0.27% to 4.23%. In all patients, the subsequent post-intra-arterial RB1 VAF was lower than baseline (0.0%-0.17%). At 4 months (2 months after IAC completion), the ctDNA consistently was negative in the patients who demonstrated clinically inactive intraocular disease.
UNASSIGNED: In this small cohort, a decremental decrease in ctDNA RB1 VAF was found after IAC, suggesting that relative VAF changes could be a biomarker of treatment response.

UNASSIGNED: Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions.
UNASSIGNED: Retrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course.
UNASSIGNED: Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions.
UNASSIGNED: The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease.
UNASSIGNED: Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features.
UNASSIGNED: RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma.
UNASSIGNED: Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.

Single-cell sequencing has gained popularity in recent years. Despite its numerous applications, single-cell DNA sequencing data is highly error-prone due to technical biases arising from uneven sequencing coverage, allelic dropout, and amplification error. With these artifacts, the identification of somatic genomic variants becomes a challenging task, and over the years, several methods have been developed explicitly for this type of data. Single-cell variant callers implement distinct strategies, make different use of the data, and typically result in many discordant calls when applied to real data. Here, we review current approaches for single-cell variant calling, emphasizing single nucleotide variants. We highlight their potential benefits and shortcomings to help users choose a suitable tool for their data at hand.

Diffuse glioma is a highly heterogeneous central nervous system tumor that is refractory to conventional therapy. Residual glioma cells escape from surgery and chemoradiotherapy, leading to lethal recurrence. Understanding the molecular mechanism of this recurrence process is critical to the development of successful therapies. Here, we analyzed whole-exome sequencing (WES) data of 97 paired primary and recurrent samples from 46 patients with glioma via a uniform pipeline. Clonality and phylogenetic analyses revealed that branching evolution was widespread in the recurrent process of gliomas. Recurrent tumors continued to evolve independently with chemoradiotherapy and harbored multiple recurrence-selected genetic alterations, such as amplification of PPFIBP1, PDE4DIP, and KRAS, deletion of TNFRSF14, DCC, CDKN2A, and MSH6, and mutations in ATRX, ARID1A, KEL, TP53, MSH6, and KMT2B. Meanwhile, truncal variants within partial driver genes were identified among primary and recurrent gliomas, suggesting that they might be ideal therapeutic targets. Intriguingly, the immunogenicity of recurrent gliomas did not increase significantly compared to the primary tumors. Genomic analysis of recurrent gliomas provided an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors.

UNASSIGNED: Clonal hematopoiesis of indeterminate potential (CHIP) is a novel cardiovascular disease (CVD) risk factor in individuals without acute myeloid leukemia (AML).
UNASSIGNED: The aim of this study was to examine the association between mutations associated with CHIP (CHIP-related mutations) identified in patients at AML diagnosis and the risk for cardiovascular events (CVEs).
UNASSIGNED: This was a retrospective cohort study of 623 patients with AML treated between 2015 and 2018 who underwent DNA analysis. Cause-specific hazard regression models were used to study the associations between pathogenic mutations in common CHIP-related genes (DNMT3A, TET2, ASXL1, JAK2, TP53, SRSF2, and SF3B1) and the rate of CVEs (heart failure hospitalization, acute coronary syndrome, coronary artery revascularization, ischemic stroke, venous thromboembolism, and CVD death) and between CVE development and all-cause mortality.
UNASSIGNED: Patients were 64.6 ± 15.3 years of age, 265 (42.5%) were women, and 63% had at least 1 CHIP-related mutation. Those with CHIP-related mutations were older (69.2 ± 12.3 vs 56.6 ± 16.6 years; P < 0.001) and had a greater prevalence of CVD risk factors and CVD history. In adjusted analysis, the presence of any CHIP-related mutation was associated with a higher rate of CVEs (HR: 1.74; 95% CI: 1.03-2.93; P = 0.037) among intensively treated patients (anthracycline based) but not the whole cohort (HR: 1.26; 95% CI: 0.81-1.97; P = 0.31). TP53 (HR: 4.18; 95% CI: 2.07-8.47; P < 0.001) and ASXL1 (HR: 2.37; 95% CI: 1.21-4.63; P = 0.012) mutations were associated with CVEs among intensively treated patients. Interval development of CVEs was associated with all-cause mortality (HR: 1.99; 95% CI: 1.45-2.73; P < 0.001).
UNASSIGNED: Among patients with AML treated with intensive chemotherapy, mutations in CHIP-related genes were associated with an increased risk for developing incident CVEs after AML diagnosis.

Next Generation Sequencing (NGS) has dramatically improved the flexibility and outcomes of cancer research and clinical trials, providing highly sensitive and accurate high-throughput platforms for large-scale genomic testing. In contrast to whole-genome (WGS) or whole-exome sequencing (WES), targeted genomic sequencing (TS) focuses on a panel of genes or targets known to have strong associations with pathogenesis of disease and/or clinical relevance, offering greater sequencing depth with reduced costs and data burden. This allows targeted sequencing to identify low frequency variants in targeted regions with high confidence, thus suitable for profiling low-quality and fragmented clinical DNA samples. As a result, TS has been widely used in clinical research and trials for patient stratification and the development of targeted therapeutics. However, its transition to routine clinical use has been slow. Many technical and analytical obstacles still remain and need to be discussed and addressed before large-scale and cross-centre implementation. Gold-standard and state-of-the-art procedures and pipelines are urgently needed to accelerate this transition. In this review we first present how TS is conducted in cancer research, including various target enrichment platforms, the construction of target panels, and selected research and clinical studies utilising TS to profile clinical samples. We then present a generalised analytical workflow for TS data discussing important parameters and filters in detail, aiming to provide the best practices of TS usage and analyses.