Joni Van der Meulen

An important step toward clinical implementation of (epi-)genomic assays on liquid biopsies is their validation on identical samples within and across laboratories. For these validation studies, there is a need for cell-free DNA (cfDNA) samples with defined tumor fractions and (epi-)genomic aberrations. However, the amount of circulating cfDNA isolated from patient samples is often limited, especially in pediatric cases. Additionally, patient samples contain a high degree of variability in cfDNA yield and tumor fraction. Several commercial artificial cfDNA products are available for validation studies, however their use is restricted to specific assays, aberrations and/or tumor entities. Alternatively, artificial cfDNA samples can be produced by fragmenting genomic DNA to mimic highly fragmented cfDNA derived from both tumor and healthy blood, followed by mixing artificial tumoral and healthy cfDNA at defined fractions. In this study, we compared native cfDNA with artificial cfDNA generated by three different fragmentation methods, including sonication and two enzymatic digestions using micrococcal nuclease and double-stranded deoxyribonuclease (dsDNase). We assessed fragment length profiles, end motifs and nucleosome occupancy patterns from shallow whole-genome sequencing data, as well as coverage profiles from targeted panel sequencing, together with a small-scale mixing experiment of tumor and healthy cell derived artificial cfDNA. Although sonication remains a convenient high-throughput approach to generate artificial cfDNA for certain downstream applications, enzymatic fragmentation, particularly the dsDNase-based method, more faithfully reproduced native cfDNA characteristics.

Context: Nodular fasciitis (NF), myositis ossificans/fibro‐osseous pseudotumor of the digits (MO/FP) and aneurysmal bone cyst (ABC) are grouped under the category of ‘ USP6 ‐associated neoplasms’ (UANs) due to their shared characteristic of an underlying USP6 rearrangement. Objective: Our aim in this study is to investigate the diversity of USP6 rearrangements in UANs. Design: We performed a clinicopathological and molecular investigation using targeted RNA sequencing on 124 cases of NF, 19 cases of MO/PF and 32 cases of ABC. Additionally, NF cases were scored for the presence of typical morphological features to assess their correlation with the detected USP6 fusion partner. Results: In 85.4% of NF cases (88/103), we identified a USP6 rearrangement, with 46.6% exhibiting the classic MYH9::USP6 fusion and 53.4% displaying non‐ MYH9::USP6 fusions. Notably, classic histological features of NF strongly correlated with the presence of the MYH9::USP6 fusion, while specific NF locations (e.g. intramuscular, intra‐articular and intradermal) were significantly associated with non‐ MYH9 fusions. This study identified 22 novel USP6 fusion partners, demonstrating the fusion diversity within UANs. Additionally, there was minimal overlap in USP6 fusion partners between different UAN types. Conclusions: A significant entity‐ and location‐specific USP6 fusion partner diversity was revealed among the UAN members. This supports the hypothesis that this diversity may be linked to the cell of origin. Additionally, molecular analysis can be valuable in diagnosing non‐classical NF.

Aims: Atypical spindle cell lipomatous tumour (ASLT) and atypical pleomorphic lipomatous tumour (APLT) have been grouped together under the umbrella designation atypical spindle cell/pleomorphic lipomatous tumour (ASPLT) in the 2020 edition of the World Health Organization (WHO) Classification of Soft Tissue and Bone Tumours. They are thought to exist on a morphologic spectrum and share similar clinicopathologic and biological characteristics. The aim of this study was to further explore the genetic background of ASLTs and APLTs by employing DNA‐based next‐generation sequencing and immunohistochemistry, with a specific focus on the TP53 gene.Methods and results: Using DNA‐based NGS and immunohistochemistry, TP53 alterations were identified in 20 out of 21 APLT cases (95%). This is in contrast to the ASLT cases, in which no TP53 alterations could be observed. Among APLT cases with an abnormal p53 immunohistochemical profile and successful DNA NGS testing, 92% (12 of 13 cases) harboured a TP53 alteration.Conclusions: APLTs predominantly harbour a TP53 alteration in contrast to ASLT cases. Our findings support the classification of APLT as a distinct (sub)entity within a spectrum that overlaps with ASLT, and it remains to be determined whether the broader term ‘ASPLT’ will hold up. Furthermore, p53 immunostaining proved to be a potentially valuable diagnostic tool, aiding pathologists in differentiating between ASLT and APLT.

Somatic multigene analysis by next‐generation sequencing (NGS) is routinely integrated in medical oncology for clinical decision‐making. However, with the fast‐growing number of recommended and required genes as well as pan‐cancer biomarkers, small panels have become vastly insufficient. Comprehensive genomic profiling (CGP) is, thus, required to screen for clinically relevant markers. In this multicentric study, we report on an extensive analysis across seven centers comparing the results of the novel OncoDEEP CGP assay with the diagnostically validated TruSight Oncology 500 (TSO500) kit on 250 samples. Overall concordance was 90% for clinically relevant gene variants and >96% for more complex biomarkers. Agreement for fusion detection was 94% for the 11 overlapping clinically actionable driver genes. The higher coverage uniformity of OncoDEEP compared to TSO500 allows users to pool more samples per sequencing run. Tertiary data analysis, including reporting, is integrated in the OncoDEEP solution, whereas this is an add‐on for TSO500. Finally, we showed that, analytically, the OncoDEEP panel performs well, thereby advocating its use for CGP of solid tumors in diagnostic laboratories, providing an all‐in‐one solution for optimal patient management.

Ovarian cancer is the most lethal gynecologic malignancy, mainly due to late-stage diagnosis, frequent recurrences, and eventually therapy resistance. To identify potentially actionable genetic variants, sequencing data of 351 Belgian ovarian cancer patients were retrospectively captured from electronic health records. The cohort included 286 (81%) patients with high-grade serous ovarian cancer, 17 (5%) with low-grade serous ovarian cancer, and 48 (14%) with other histotypes. Firstly, an overview of the prevalence and spectrum of the BRCA1/2 variants highlighted germline variants in 4% (11/250) and somatic variants in 11% (37/348) of patients. Secondly, application of a multi-gene panel in 168 tumors revealed a total of 214 variants in 28 genes beyond BRCA1/2 with a median of 1 (IQR, 1–2) genetic variant per patient. The ten most often altered genes were (in descending order): TP53, BRCA1, PIK3CA, BRCA2, KRAS, ERBB2 (HER2), TERT promotor, RB1, PIK3R1 and PTEN. Of note, the genetic landscape vastly differed between the studied histotypes. Finally, using ESCAT the clinical evidence of utility for every genetic variant was scored. Only BRCA1/2 pathogenic variants were classified as tier-I. Nearly all patients (151/168; 90%) had an ESCAT tier-II variant, most frequently in TP53 (74%), PIK3CA (9%) and KRAS (7%). In conclusion, our findings imply that although only a small proportion of genetic variants currently have direct impact on ovarian cancer treatment decisions, other variants could help to identify novel (personalized) treatment options to address the poor prognosis of ovarian cancer, particularly in rare histotypes.