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Two decades after the genomics revolution, oncology is rapidly transforming into a genome-driven discipline, yet routine cancer diagnostics is still mainly microscopy based, except for tumor type-specific predictive molecular tests. Pathology laboratories struggle to quickly validate and adopt biomarkers identified by genomics studies of new targeted therapies. Consequently, clinical implementation of newly approved biomarkers suffers substantial delays, leading to unequal patient access to these therapies. Whole-genome sequencing (WGS) can successfully address these challenges by providing a stable molecular diagnostic platform that allows detection of a multitude of genomic alterations in a single cost-efficient assay and facilitating rapid implementation, as well as by the development of new genomic biomarkers. Recently, the Whole-genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE) study demonstrated that WGS is a feasible and clinically valid technique in routine clinical practice with a turnaround time of 11 workdays. As a result, WGS was successfully implemented at the Netherlands Cancer Institute as part of routine diagnostics in January 2021. The success of implementing WGS has relied on adhering to a comprehensive protocol including recording patient information, sample collection, shipment and storage logistics, sequencing data interpretation and reporting, integration into clinical decision-making and data usage. This protocol describes the use of fresh-frozen samples that are necessary for WGS but can be challenging to implement in pathology laboratories accustomed to using formalin-fixed paraffin-embedded samples. In addition, the protocol outlines key considerations to guide uptake of WGS in routine clinical care in hospitals worldwide., (© 2023. Springer Nature Limited.)

Purpose: Genome sequencing (GS) enables comprehensive molecular analysis of tumors and identification of hereditary cancer predisposition. According to guidelines, directly determining pathogenic germline variants (PGVs) requires pretest genetic counseling, which is cost-ineffective. Referral for genetic counseling based on tumor variants alone could miss relevant PGVs and/or result in unnecessary referrals., Methods: We validated GS for detection of germline variants and simulated 3 strategies using paired tumor-normal GS data of 937 metastatic patients. In strategy-1, genetic counseling before tumor testing allowed direct PGV analysis. In strategy-2 and -3, germline testing and referral for post-test genetic counseling is based on tumor variants using Dutch (strategy-2) or Europen Society for Medical Oncology (ESMO) Precision Medicine Working Group (strategy-3) guidelines., Results: In strategy-1, PGVs would be detected in 50 patients (number-needed-to counsel; NTC = 18.7). In strategy-2, 86 patients would have been referred for genetic counseling and 43 would have PGVs (NTC = 2). In strategy-3, 94 patients would have been referred for genetic counseling and 32 would have PGVs (NTC = 2.9). Hence, 43 and 62 patients, respectively, were unnecessarily referred based on a somatic variant., Conclusion: Both post-tumor test counseling strategies (2 and 3) had significantly lower NTC, and strategy-2 had the highest PGV yield. Combining pre-tumor test mainstreaming and post-tumor test counseling may maximize the clinically relevant PGV yield and minimize unnecessary referrals., Competing Interests: Conflict of Interest Kim Monkhorst reports research grants from AstraZeneca and speakers’ fees from Merck Sharp & Dohme, Roche, AstraZeneca, and Benecke. Kim Monkhorst received consultancy fees from Pfizer, Bristol-Myers Squibb, Roche, Merck Sharp & Dohme, AbbVie, AstraZeneca, Diaceutics, Eli Lilly, Bayer, and Boehringer Ingelheim and nonfinancial support from Roche, Takeda, Pfizer, Personal Genome Diagnostics, and DELFi. Edwin Cuppen reports consultancy fees and support for attending meetings and traveling from Illumina. Emile E. Voest is member of the supervisory board of Hartwig Medical Foundation. Gerrit A. Meijer is cofounder and a board member (CSO) of CRCbioscreen BV, he has a research collaboration with CZ Health Insurances (cash matching to ZonMW grant), and he has research collaborations with Exact Sciences, Sysmex, Sentinel Ch. SpA, Personal Genome Diagnostics (PGDX), and DELFi; these companies provide materials, equipment, and/or sample/genomic analyses. Gerrit A. Meijer is an advisory board member of “Missie Tumor Onbekend.” All other authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: In ∼3%-5% of patients with metastatic disease, tumor origin remains unknown despite modern imaging techniques and extensive pathology work-up. With long diagnostic delays and limited and ineffective therapy options, the clinical outcome of patients with cancer of unknown primary (CUP) remains poor. Large-scale genome sequencing studies have revealed that tumor types can be predicted based on distinct patterns of somatic variants and other genomic characteristics. Moreover, actionable genomic events are present in almost half of CUP patients. This study investigated the clinical value of whole genome sequencing (WGS) in terms of primary tumor identification and detection of actionable events, in the routine diagnostic work-up of CUP patients., Patients and Methods: A WGS-based tumor type 'cancer of unknown primary prediction algorithm' (CUPPA) was developed based on previously described principles and validated on a large pan-cancer WGS database of metastatic cancer patients (>4000 samples) and 254 independent patients, respectively. We assessed the clinical value of this prediction algorithm as part of routine WGS-based diagnostic work-up for 72 CUP patients., Results: CUPPA correctly predicted the primary tumor type in 78% of samples in the independent validation cohort (194/254 patients). High-confidence predictions (>95% precision) were obtained for 162/254 patients (64%). When integrated in the diagnostic work-up of CUP patients, CUPPA could identify a primary tumor type for 49/72 patients (68%). Most common diagnoses included non-small-cell lung (n = 7), gastroesophageal (n = 4), pancreatic (n = 4), and colorectal cancer (n = 3). Actionable events with matched therapy options in clinical trials were identified in 47% of patients., Conclusions: Genome-based tumor type prediction can predict cancer diagnoses with high accuracy when integrated in the routine diagnostic work-up of patients with metastatic cancer. With identification of the primary tumor type in the majority of patients and detection of actionable events, WGS is a valuable diagnostic tool for patients with CUP., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 (<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 Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

With more than 70 different histological sarcoma subtypes, accurate classification can be challenging. Although characteristic genetic events can largely facilitate pathological assessment, large-scale molecular profiling generally is not part of regular diagnostic workflows for sarcoma patients. We hypothesized that whole genome sequencing (WGS) optimizes clinical care of sarcoma patients by detection of diagnostic and actionable genomic characteristics, and of underlying hereditary conditions. WGS of tumor and germline DNA was incorporated in the diagnostic work-up of 83 patients with a (presumed) sarcomas in a tertiary referral center. Clinical follow-up data were collected prospectively to assess impact of WGS on clinical decision making. In 12/83 patients (14%), the genomic profile led to revision of cancer diagnosis, with change of treatment plan in eight. All twelve patients had undergone multiple tissue retrieval procedures and immunohistopathological assessments by regional and expert pathologists prior to WGS analysis. Actionable biomarkers with therapeutic potential were identified for 30/83 patients. Pathogenic germline variants were present in seven patients. In conclusion, unbiased genomic characterization with WGS identifies genomic biomarkers with direct clinical implications for sarcoma patients. Given the diagnostic complexity and high unmet need for new treatment opportunities in sarcoma patients, WGS can be an important extension of the diagnostic arsenal of pathologists.

Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable 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 single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0%; EGFR 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 (human papillomavirus), an accuracy of 100% compared with standard testing. In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 John Wiley & Sons Ltd.)

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.

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.