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The current increase in number and diversity of targeted anticancer agents poses challenges to the logistics and timeliness of molecular diagnostics (MolDx), resulting in underdiagnosis and treatment. Whole-genome sequencing (WGS) may provide a sustainable solution for addressing current as well as future diagnostic challenges. The present study therefore aimed to prospectively assess feasibility, validity, and value of WGS in routine clinical practice. WGS was conducted independently of, and in parallel with, standard of care (SOC) diagnostics on routinely obtained tumor samples from 1,200 consecutive patients with metastatic cancer. Results from both tests were compared and discussed in a dedicated tumor board. From 1,200 patients, 1,302 samples were obtained, of which 1,216 contained tumor cells. WGS was successful in 70% (854/1,216) of samples with a median turnaround time of 11 days. Low tumor purity (lt;20%) was the main reason for not completing WGS. WGS identified 99.2% and SOC MolDx 99.7% of the total of 896 biomarkers found in genomic regions covered by both tests. Actionable biomarkers were found in 603/848 patients (71%). Of the 936 associated therapy options identified by WGS, 343 were identified with SOC MolDx (36.6%). Biomarker-based therapy was started in 147 patients. WGS revealed 49 not previously identified pathogenic germline variants. Fresh-frozen, instead of formalin-fixed and paraffin-embedded, sample logistics were easily adopted as experienced by the professionals involved. WGS for patients with metastatic cancer is well feasible in routine clinical practice, successfully yielding comprehensive genomic profiling for the vast majority of patients. © 2022 The Authors. The Journal of Pathology published by John Wileyamp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Introduction: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions.Methods: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci.Results: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci.Conclusions: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC. (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc.

Background Small-intestinal neuroendocrine tumours (SI-NETs) represent a heterogeneous group of rare tumours. In recent years, basic research in SI-NETs has attempted to unravel the molecular events underlying SI-NET tumorigenesis. Aim We aim to provide an overview of the current literature regarding prognostic and predictive molecular factors in patients with SI-NETs. Method A PubMed search was conducted on (epi)genetic prognostic factors in SI-NETs from 2000 until 2019. Results The search yielded 1522 articles of which 20 reviews and 35 original studies were selected for further evaluation. SI-NETs are mutationally quiet tumours with a different genetic make-up compared to pancreatic NETs. Loss of heterozygosity at chromosome 18 is the most frequent genomic aberration (44–100%) followed by mutations of CDKN1B in 8%. Prognostic analyses were performed in 16 studies, of which 8 found a significant (epi)genetic association for survival or progression. Loss of heterozygosity at chromosome 18, gains of chromosome 4, 5, 7, 14 and 20p, copy gain of the SRC gene and low expression of RASSF1A and P16 were associated with poorer survival. In comparison with genetic mutations, epigenetic alterations are significantly more common in SI-NETs and may represent more promising targets in the treatment of SI-NETs. Conclusion SI-NETs are mutationally silent tumours. No biomarkers have been identified yet that can easily be adopted into current clinical decision making. SI-NETs may represent a heterogeneous disease and larger international studies are warranted to translate molecular findings into precision oncology.

Whole genome sequencing (WGS) using fresh frozen tissue and matched blood samples from cancer patients is becoming in reach as the most complete genetic tumor test. With a trend towards the availability of small biopsies and the need to screen an increasing number of (complex) biomarkers, the use of a single all-inclusive test is preferred over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content.Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall) (98.5%) and precision (positive predictive value) (97.8%) for detection of somatic SNV and indels (across 22 genes), and high concordance for detection of gene amplification (97.0%, EGRF and MET) as well as somatic complete loss (100%, CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (HPV) an accuracy of 100% compared to standard testing.In conclusion, whole genome sequencing has a >95% sensitivity and precision compared to routinely used DNA techniques in diagnostics and all relevant mutation types can be detected reliably in a single assay.

Background ‘Precision oncology’ can ensure the best suitable treatment at the right time by tailoring treatment towards individual patient and comprehensive tumour characteristics. In current molecular pathology, diagnostic tests which are part of the standard of care (SOC) only cover a limited part of the spectrum of genomic changes, and often are performed in an iterative way. This occurs at the expense of valuable patient time, available tissue sample, and interferes with ‘first time right’ treatment decisions. Whole Genome Sequencing (WGS) captures a near complete view of genomic characteristics of a tumour in a single test. Moreover, WGS facilitates faster implementation of new treatment relevant biomarkers. At present, WGS mainly has been applied in study settings, but its performance in a routine diagnostic setting remains to be evaluated. The WIDE study aims to investigate the feasibility and validity of WGS-based diagnostics in clinical practice. Methods 1200 consecutive patients in a single comprehensive cancer centre with (suspicion of) a metastasized solid tumour will be enrolled with the intention to analyse tumour tissue with WGS, in parallel to SOC diagnostics. Primary endpoints are (1) feasibility of implementation of WGS-based diagnostics into routine clinical care and (2) clinical validation of WGS by comparing identification of treatment-relevant variants between WGS and SOC molecular diagnostics. Secondary endpoints entail (1) added clinical value in terms of additional treatment options and (2) cost-effectiveness of WGS compared to SOC diagnostics through a Health Technology Assessment (HTA) analysis. Furthermore, the (3) perceived impact of WGS-based diagnostics on clinical decision making will be evaluated through questionnaires. The number of patients included in (experimental) therapies initiated based on SOC or WGS diagnostics will be reported with at least 3 months follow-up. The clinical efficacy is beyond the scope of WIDE. Key performance indicators will be evaluated after every 200 patients enrolled, and procedures optimized accordingly, to continuously improve the diagnostic performance of WGS in a routine clinical setting. Discussion WIDE will yield the optimal conditions under which WGS can be implemented in a routine molecular diagnostics setting and establish the position of WGS compared to SOC diagnostics in routine clinical care.

Aims To investigate the clinicopathological significance of driver mutations in metastatic well-differentiated small intestine neuroendocrine tumours (SI-NETs). Methods and results Whole genome sequencing (WGS) of 35 metastatic SI-NETs and next-generation sequencing (NGS) of eight metastatic SI-NETs were performed. Biopsies were obtained between 2015 and 2019. Tumours were classified according to the 2019 World Health Organization classification. WGS included assessment of somatic mutations in all cancer-related driver genes, the tumour mutational burden (TMB), and microsatellite status. NGS entailed a cancer hotspot panel of 58 genes. Our cohort consisted of 21% grade 1, 60% grade 2 and 19% grade 3 SI-NETs. Driver mutations were identified in ~50% of SI-NETs. In total, 27 driver mutations were identified, of which 74% were in tumour suppressor genes (e.g. TP53, RB1, and CDKN1B) and 22% were in proto-oncogenes (e.g. KRAS, NRAS, and MET). Allelic loss of chromosome 18 (63%), complete loss of CDKN2A and CDKN1B (both 6%) and CDKN1B mutations (9%) were most common. Potential targetable genetic alterations were detected in 21% of metastasised SI-NETs. All tumours were microsatellite-stable and showed low TMBs (median 1.10; interquartile range 0.87-1.35). The Ki67 proliferation index was significantly associated with the presence of driver mutations (P = 0.015). Conclusion Driver mutations occur in 50% of metastasised SI-NETs, and their presence is associated with a high Ki67 proliferation index. The identification of targetable mutations make these patients potentially eligible for targeted therapy.

11540 Background: With more than 70 different histological subtypes, accurate classification sarcomas is challenging. Although pathognomonic genetic events aid accurate classification, large-scale molecular profiling is generally not incorporated in regular diagnostic workflows for sarcoma patients. We hypothesized that whole genome sequencing (WGS) optimizes clinical care of sarcoma patients by detection of pathognomonic and actionable variants, and of underlying hereditary conditions. Methods: WGS of tumor and germline DNA was incorporated in the diagnostic work-up of 83 patients with a (presumed) sarcoma as part of the WIDE (Whole genome sequencing Implementation in standard Diagnostics for Every cancer patient) study in a tertiary referral center. WGS results were reported back to the pathologist and treating clinician. Clinical follow-up data were collected prospectively to assess impact of WGS on clinical decision making. Results: WGS analysis had impact on multiple levels. First, in 14% of cases (12/83 patients), the genomic profile led to a revision of the diagnosis (table). All patients had undergone multiple diagnostic procedures (mean number: 4) and pathologist assessments (mean: 6) before WGS analysis was performed. Secondly, actionable biomarkers with therapeutic potential were detected for 36/83 patients and finally, 8 pathogenic germline variants were present. Taken together, WGS had implications for clinical decision making in 52% of patients with (presumed) sarcomas. Conclusions: WGS is an important extension of the diagnostic arsenal of pathologists and has contributed to change of care in 52% of patients with sarcomas. Given the diagnostic complexity and high unmet need for new treatment opportunities in sarcomas we advocate the use of WGS for sarcoma patients early in the disease course. Clinical trial information: NL68609.031.18. [Table: see text]

3013 Background: In the next few years numerous drugs will be approved for defined genomic targets, most of these in a tumor agnostic manner. Identifying patients who can benefit from this is critical for the future success of precision medicine, ideally using a single comprehensive test to detect all possible biomarkers. The WIDE study (WGS Implementation in standard cancer Diagnostics for Every cancer patient) aimed to evaluate the feasibility, clinical validity (primary endpoints) and added value (secondary endpoint) of clinical grade Whole Genome Sequencing (cWGS) in routine clinical practice. Methods: cWGS was prospectively performed on 1,200 consecutive patients with (suspected) metastatic cancer. Tumor material was obtained during routine clinical procedures for both Standard-Of-Care (SOC) and cWGS. Next to securing a high quality specimen for SOC diagnostics, multiple frozen sections per patient were evaluated to identify the sample most suitable for WGS. cWGS was conducted independently of, but in parallel with SOC diagnostics, which included SOC molecular diagnostics (Moldx) for 48% of patients. cWGS and MolDx results were compared and discussed in a dedicated tumor board. Additional tests for resolving discordances were applied when needed. Results: cWGS was successfully performed in 69% (841/1217) of samples with a technical success rate of 97% (841/871). An insufficient amount of tumor cells ( < 20%) was the main reason for not completing cWGS (25%, 310/1217). cWGS turn-around-time (TAT), due to continuous improvements to the clinical procedure and cWGS pipeline over the course of the study, decreased to 10 working days. A total of 856 genomic biomarkers identified by SOC MolDx could be compared to cWGS results. Initial analyses of discordances revealed an error rate of 2.1% (18/856) for cWGS compared to a 1.0% (8/856) error rate for SOC Moldx. After optimizing cWGS and SOC pipelines based on these findings, error rates dropped to 0.6% (5/856) and 0.7% (6/856) for cWGS and SOC MolDx, respectively. Overall, cWGS identified clinically actionable (routine practice and experimental) biomarkers in 71% of all patients tested. Compared to SOC MolDx, cWGS identified one or more additional clinically actionable biomarkers in 54% (446/832) of patients. Interestingly, in patients who were not tested by SOC MolDx, actionable variants were identified by cWGS in 54% (153/282). Conclusions: The WIDE study has shown that cWGS is feasible in routine clinical practice in a comprehensive cancer center setting, using tumor material obtained during routine procedures. Furthermore, cWGS showed added value by identifying one or more additional clinically actionable biomarkers in 54% of patients including patients who had not received SOC Moldx. These outcomes have led to the successful adoption of cWGS at the Netherlands Cancer Institute as part of routine care, which will further facilitate precision medicine for cancer patients.

Background Advances in DNA sequencing technology have strongly reduced costs of Whole Genome Sequencing (WGS) and have made it possible to perform WGS on tumor biopsies within 2 to 3 weeks. Despite its great potential, the value of WGS has so far only been addressed in retrospective or small prospective studies. We have retrospectively shown in > 2,400 metastatic cancer patients that in 31% an approved biomarker for a targeted treatment could be identified. Of these biomarkers, 58% were not targets for standard-of-care (SoC) treatment but suggested off-label use or clinical study eligibility. The potential of WGS in a routine diagnostic setting is explored in the WIDE study and will address feasibility, clinical validity and added value of WGS. Methods WGS will be performed on a prospective cohort of 1200 patients with (suspicion of) stage IV metastatic cancer including all solid tumor types. WGS is conducted at the Hartwig Medical Foundation independently of, and in parallel with, SoC diagnostics at the Netherland Cancer Institute. Results are discussed in a tumor board to assess the value of WGS findings for treatment decision and clinical study eligibility. In addition, cost-effectiveness will be evaluated and all participating treating physicians will be asked to what extent WGS has aided their decisions making. Results With an accrual rate of 50-75 patients/month starting end of Q2 2019, results of the first 200 patients will be presented. Data from 25 retrospective cases (all with WGS, and 13 with parallel SoC MDx) indicated targets for standard targeted treatment in 5 patients (EGFR, ERBB2 and ROS1 inhibitors) and were identified both WGS and SoC MDx. In 19 patients, WGS found potential clinical study eligibility targets (including PI3K/mTOR, CDK4/6, MEK, FGFR1, MDM2, PARP and checkpoint inhibitors), whereas SoC MDx identified only 6 of these patients as potentially eligible. No relevant DNA aberration was identified for 5 patients. Conclusions The WIDE study will provide quantifiable data regarding the feasibility, clinical validity and added value of WGS analysis for patients with metastatic cancer in a routine clinical diagnostic setting. Legal entity responsible for the study Netherlands Cancer Institute. Funding Illumina. Disclosure All authors have declared no conflicts of interest.