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Circulating tumor DNA (ctDNA) detection has multiple promising applications in oncology, but the road toward implementation in clinical practice is unclear. We aimed to support the implementation process by exploring potential future pathways of ctDNA testing. To do so, we studied four ctDNA-testing applications in two cancer types and elicited opinions from 30 ctDNA experts in the Netherlands. Our results showed that the current available evidence differed per application and cancer type. Tumor profiling and monitoring treatment response were found most likely to be implemented in non-small cell lung cancer (NSCLC) within 5 years. For colorectal cancer, applications of ctDNA testing were found to be at an early stage in the implementation process. Demonstrating clinical utility was found a key aspect for successful implementation, but there was no consensus regarding the evidence requirements. The next step toward implementation is to define how clinical utility of biomarkers should be evaluated. Finally, these data indicate that specific challenges for each clinical application and tumor type should be appropriately addressed in a deliberative process involving all stakeholders to ensure implementation of ctDNA testing and timely access for patients., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Structural variants (SVs) caused by chromosomal rearrangements in common fragile sites or long interspersed nuclear element (LINE) retrotranspositions are highly prevalent in colorectal cancer. However, methodology for the targeted detection of these SVs is lacking. This article reports the use of formalin-fixed paraffin-embedded targeted-locus capture (FFPE-TLC) sequencing as a novel technology for the targeted detection of tumor-specific SVs. Analysis of 29 FFPE colorectal tumor samples and 8 matched normal samples revealed tumor-specific SVs in 24 patients (83%), with a median of 2 SVs per patient (range, 1 to 21). A total of 104 SVs were found in the common fragile site-associated genes MACROD2, PRKN, FHIT, and WWOX in 18 patients (62%), and 39 SVs caused by three LINE transposable elements were found in 15 patients (52%). Tumor specificity of SVs was independently verified by Droplet Digital PCR of tumor tissue DNA, and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. It was concluded that FFPE-TLC sequencing enables the detection of tumor-specific SVs caused by chromosomal rearrangements and LINE retrotranspositions in FFPE tissue. Therefore, FFPE-TLC sequencing facilitates the investigation of the biological and clinical effects of SVs using FFPE material from (retrospective) cohorts of cancer patients and has potential clinical applicability in the detection of SV biomarkers in the routine molecular diagnostics setting., Competing Interests: Disclosure Statement C.R.-A. declares nonfinancial support from Personal Genome Diagnostics and Cergentis BV. E.S., J.S., and H.F. are employees of Cergentis BV (a Solvias company), the inventor and owner of the patents on FFPE-TLC technology. S.L. reports nonfinancial support from Cergentis BV and a patent pending. E.J.v.B. reports nonfinancial support from Cergentis BV. D.v.d.B. has provided lectures, expert testimony, and advisory board presence to Roche Diagnostics, all outside the submitted work and all financial supports transferred to institute. G.A.M. is co-founder and board member (CSO) of CRCbioscreen BV; has had research collaboration with CZ Health Insurances (cash matching to ZonMW grant); has had research collaborations with Exact Sciences, Sysmex, Sentinel Ch SpA, Personal Genome Diagnostics, DELFi, and HMF (these companies provide materials, equipment and/or sample/genomic analyses); and has several patents pending/issued. S.A. reports public–private partnership consortia grants in collaboration with Cergentis BV, Olink Proteomics AB, and Quanterix Corporation and has a patent pending. R.J.A.F. reports public–private partnership consortia grants in collaboration with Cergentis BV; reports public–private partnership consortia grants and nonfinancial support in collaboration with Personal Genome Diagnostics, Delfi, Natera, and Merck BV outside the submitted work; and has several patents pending., (Copyright © 2024. Published by Elsevier Inc.)

Circulating tumor DNA (ctDNA) is a promising new biomarker with multiple potential applications in cancer care. Estimating total cost of ctDNA testing is necessary for reimbursement and implementation, but challenging because of variations in workflow. We aimed to develop a micro-costing framework for consistent cost calculation of ctDNA testing. First, the foundation of the framework was built, based on the complete step-wise diagnostic workflow of ctDNA testing. Second, the costing method was set up, including costs for personnel, materials, equipment, overhead, and failures. Third, the framework was evaluated by experts and applied to six case studies, including PCR-, mass spectrometry-, and next-generation sequencing-based platforms, from three Dutch hospitals. The developed ctDNA micro-costing framework includes the diagnostic workflow from blood sample collection to diagnostic test result. The framework was developed from a Dutch perspective and takes testing volume into account. An open access tool is provided to allow for laboratory-specific calculations to explore the total costs of ctDNA testing specific workflow parameters matching the setting of interest. It also allows to straightforwardly assess the impact of alternative prices or assumptions on the cost per sample by simply varying the input parameters. The case studies showed a wide range of costs, from €168 to €7638 ($199 to $9124) per sample, and generated information. These costs are sensitive to the (coverage of) platform, setting, and testing volume., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening., Competing Interests: Declaration of interests M.G. Best, R.J.A.N., and T.W. are inventors on relevant patent applications (PCT/NL2011/050518 and PCT/NL2018/050110). R.J.A.N. and T.W. are shareholders of Illumina, Inc. M.H. is chief formulation officer at Nurish.Me, Inc., and Camelina Sun LLC and has equity in those companies (whose business activities are unrelated to the present work). D.M.P. and D.K.L. are shareholders of ExBiome BV., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stage II-IIIA nonsmall cell lung cancer (NSCLC) patients receive adjuvant chemotherapy after surgery as standard-of-care treatment, even though only approximately 5.8% of patients will benefit. Identifying patients with minimal residual disease (MRD) after surgery using tissue-informed testing of postoperative plasma circulating cell-free tumour DNA (ctDNA) may allow adjuvant therapy to be withheld from patients without MRD. However, the detection of MRD in the postoperative setting is challenging, and more sensitive methods are urgently needed. We developed a method that combines variant calling and a novel ctDNA fragment length analysis using hybrid capture sequencing data. Among 36 stage II-IIIA NSCLC patients, this method distinguished patients with and without recurrence of disease in a 20 times repeated 10-fold cross validation with 75% accuracy (P = 0.0029). In contrast, using only variant calling or only fragment length analysis, no signification distinction between patients was shown (P = 0.24 and P = 0.074 respectively). In addition, a variant-level fragmentation score was developed that was able to classify variants detected in plasma cfDNA into tumour-derived or white-blood-cell-derived variants with 84% accuracy. The findings in this study may help drive the integration of various types of information from the same data, eventually leading to cheaper and more sensitive techniques to be used in this challenging clinical setting., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Tissue biopsies can be burdensome and are only effective in 10-30% of patients with metastasized non-small-cell lung cancer (mNSCLC). Next-generation sequencing (NGS) on cell-free DNA (cfDNA) might be an attractive alternative. We evaluated the costs, throughput time, and diagnostic yield of two diagnostic scenarios with tissue and cfDNA for mNSCLC patients, compared to diagnostics based on tissue biopsy alone. Data were retrieved from 209 stage IV NSCLC patients included in 10 hospitals in the Netherlands in the observational Lung cancer Early Molecular Assessment (LEMA) trial. Discrete event simulation was developed to compare three scenarios, using LEMA data as input where possible: (1) diagnostics with "tissue only"; (2) diagnostics with "cfDNA first", and subsequent tissue biopsy if required (negative for EGFR, BRAF ALK, ROS1); (3) cfDNA if tissue biopsy failed ("tissue first"). Scenario- and probabilistic analyses were performed to quantify uncertainty. In scenario 1, 84% (Credibility Interval [CrI] 70-94%) of the cases had a clinically relevant test result, compared to 93% (CrI 86-98%) in scenario 2, and 93% (CrI 86-99%) in scenario 3. The mean throughput time was 20 days (CrI 17-23) pp in scenario 1, 9 days (CrI 7-11) in scenario 2, and 19 days (CrI 16-22) in scenario 3. Mean costs were €2304 pp (CrI €2067-2507) in scenario 1, compared to €3218 (CrI €3071-3396) for scenario 2, and €2448 (CrI €2382-2506) for scenario 3. Scenarios 2 and 3 led to a reduction in tissue biopsies of 16% and 9%, respectively. In this process-based simulation analysis, the implementation of cfDNA for patients with mNSCLC resulted in faster completion of molecular profiling with more identified targets, with marginal extra costs in scenario 3.

Comprehensive molecular profiling (CMP) plays an essential role in clinical decision making in metastatic non-small-cell lung cancer (mNSCLC). Circulating tumor DNA (ctDNA) analysis provides possibilities for molecular tumor profiling. In this study, we aim to explore the additional value of centralized ctDNA profiling next to current standard-of-care protocolled tissue-based molecular profiling (SoC-TMP) in the primary diagnostic setting of mNSCLC in the Netherlands., Methods: Pretreatment plasma samples from 209 patients with confirmed mNSCLC were analyzed retrospectively using the NGS AVENIO ctDNA Targeted Kit (Roche Diagnostics, Basel, Switzerland) and compared with paired prospective pretreatment tissue-based molecular profiling from patient records. The AVENIO panel is designed to detect single-nucleotide variants, copy-number variations, insertions or deletions, and tyrosine kinase fusion in 17 genes., Results: Potentially targetable drivers were detected with SoC-TMP alone in 34.4% of patients. Addition of clonal hematopoiesis of indeterminate potential-corrected, plasma-based CMP increased this to 39.7% ( P < .001). Concordance between SoC-TMP and plasma-CMP was 86.6% for potentially targetable drivers. Clinical sensitivity of plasma-CMP was 75.2% for any oncogenic driver. Specificity and positive predictive value were more than 90% for all oncogenic drivers., Conclusion: Plasma-CMP is a reliable tool in the primary diagnostic setting, although it cannot fully replace SoC-TMP. Complementary profiling by combined SoC-TMP and plasma-CMP increased the proportion of patients who are eligible for targeted treatment., Competing Interests: Kim Monkhorst Consulting or Advisory Role: Roche Molecular Diagnostics, MSD, AstraZeneca, AbbVie, Bristol Myers Squibb, Lilly, Boehringer Ingelheim, Bayer Speakers' Bureau: Quadia Research Funding: AstraZeneca, Roche Molecular Diagnostics, Personal Genome Diagnostics Travel, Accommodations, Expenses: Takeda No other potential conflicts of interest were reported. Kim Monkhorst Consulting or Advisory Role: Roche Molecular Diagnostics, MSD, AstraZeneca, AbbVie, Bristol Myers Squibb, Lilly, Boehringer Ingelheim, Bayer Speakers' Bureau: Quadia Research Funding: AstraZeneca, Roche Molecular Diagnostics, Personal Genome Diagnostics Travel, Accommodations, Expenses: Takeda No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Background: Bio-Rad droplet-digital PCR is a highly sensitive method that can be used to detect tumor mutations in circulating cell-free DNA (cfDNA) of patients with cancer. Correct interpretation of ddPCR results is important for optimal sensitivity and specificity. Despite its widespread use, no standardized method to interpret ddPCR data is available, nor have technical artifacts affecting ddPCR results been widely studied., Methods: False positive rates were determined for 6 ddPCR assays at variable amounts of input DNA, revealing polymerase induced false positive events (PIFs) and other false positives. An in silico correction algorithm, known as the adaptive LoB and PIFs: an automated correction algorithm (ALPACA), was developed to remove PIFs and apply an adaptive limit of blank (LoB) to individual samples. Performance of ALPACA was compared to a standard strategy (no PIF correction and static LoB = 3) using data from commercial reference DNA, healthy volunteer cfDNA, and cfDNA from a real-life cohort of 209 patients with stage IV nonsmall cell lung cancer (NSCLC) whose tumor and cfDNA had been molecularly profiled., Results: Applying ALPACA reduced false positive results in healthy cfDNA compared to the standard strategy (specificity 98 vs 88%, P = 10-5) and stage IV NSCLC patient cfDNA (99 vs 93%, P = 10-11), while not affecting sensitivity in commercial reference DNA (70 vs 68% P = 0.77) or patient cfDNA (82 vs 88%, P = 0.13). Overall accuracy in patient samples was improved (98 vs 92%, P = 10-7)., Conclusions: Correction of PIFs and application of an adaptive LoB increases specificity without a loss of sensitivity in ddPCR, leading to a higher accuracy in a real-life cohort of patients with stage IV NSCLC., (© American Association for Clinical Chemistry 2021.)

Detection of KRAS, NRAS, and BRAF mutations in tumor tissue is currently used to predict resistance to treatment with anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). Liquid biopsies are minimally invasive, and cell-free circulating tumor DNA (ctDNA) mutation analyses may better represent tumor heterogeneity. This study examined the incorporation of liquid biopsy RAS/BRAF ctDNA analyses into diagnostic strategies to determine mCRC patient eligibility for anti-EGFR therapy. Tumor tissue and liquid biopsies were collected from 100 mCRC patients with liver-only metastases in a multicenter prospective clinical trial. Three diagnostic strategies incorporating droplet digital PCR ctDNA analyses were compared with routine tumor tissue RAS/BRAF mutation profiling using decision tree analyses. Tissue DNA mutations in KRAS, NRAS, and BRAF were present in 54%, 0%, and 3% of mCRC patients, respectively. A 93% concordance was observed between tissue DNA and liquid biopsy ctDNA mutations. The proportion of patients with RAS/BRAF alterations increased from 57% to 60% for diagnostic strategies that combined tissue and liquid biopsy mutation analyses. Consecutive RAS/BRAF ctDNA analysis followed by tissue DNA analysis in case of a liquid biopsy-negative result appeared to be the most optimal diagnostic strategy to comprehensively determine eligibility for anti-EGFR therapy in a cost-saving manner. These results highlight the potential clinical utility of liquid biopsies for detecting primary resistance to anti-EGFR-targeted therapies., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Multiple platforms are commercially available for the detection of circulating cell-free tumour DNA (ctDNA) from liquid biopsies. Since platforms have different input and output variables, deciding what platform to use for a given clinical or research question can be daunting. This study aimed to provide insight in platform selection criteria by comparing four commercial platforms that detect KRAS ctDNA hotspot mutations: Bio-Rad droplet digital PCR (ddPCR), BioCartis Idylla, Roche COBAS z480 and Sysmex BEAMing. Platform sensitivities were determined using plasma samples from metastatic colorectal cancer (mCRC) patients and synthetic reference samples, thereby eliminating variability in amount of plasma analysed and ctDNA isolation methods. The prevalence of KRAS nucleotide alterations was set against platform-specific breadth of target. Platform comparisons revealed that ddPCR and BEAMing detect more KRAS mutations amongst mCRC patients than Idylla and COBAS z480. Maximum sample throughput was highest for ddPCR and COBAS z480. Total annual costs were highest for BEAMing and lowest for Idylla and ddPCR. In conclusion, when selecting a platform for detection of ctDNA hotspot mutations the desired test sensitivity, breadth of target, maximum sample throughput, and total annual costs are critical factors that should be taken into consideration. Based on the results of this study, laboratories will be able to select the optimal platform for their needs.