White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer

Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA (ctDNA) alterations through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative treatment in patients with operable gastric cancer. After filtering alterations from matched white blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist .

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Victor E. Velculescu Dec 11, 2019 Primary processing of targeted NGS data for cfDNA samples was performed using Illumina CASAVA (version 1.8.2) with alignment using NovoAlign (version 3.02.12). Variant calling was performed using VariantDx.
The sequencing data from cfDNA and white blood cell samples have been deposited at the European Genome Phenome Archive (EGAS00001004105).

nature research | reporting summary
October 2018

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Clinical data Policy information about clinical studies
All manuscripts should comply with the ICMJEguidelines for publication of clinical research and a completedCONSORT checklist must be included with all submissions.

Clinical trial registration
Study protocol A total of 120 serial plasma samples from 50 patients would be needed to provide an estimate of the difference of disease recurrence of at least 45% between groups with ctDNA detected (assuming an incidence of recurrence of 75%) and not detected (assuming an incidence of recurrence of 30%) after preoperative chemotherapy in patients with resectable gastric cancer eligible for multi-modal therapy, with a 90% power at an ! = 0.05.
Plasma samples that were not collected according the described protocol were not used in the analysis.

Not applicable
Not applicable

Not applicable
Patients were eligible for this translational study if they had plasma samples available and suitable for genomic analyses, as well as histologically proven gastric adenocarcinoma, stage IB-IVA, as assessed by esophagogastroduodenoscopy and CT of the chest, abdomen, and pelvis. Patients with tumors of the gastroesophageal junction were permitted to enroll when the bulk of the tumor was predominantly located in the stomach and could therefore consist of Siewert types II (true gastroesophageal junction) and III (subcardial stomach) tumors.
Patients in this study were recruited from centers in the Netherlands (44 hospitals) that participated in the CRITICS trial (NCT00407186; Cats et al., Lancet Oncol, 2018). CRITICS trial is an investigator-initiated, open-label, multi-center, phase III study of perioperative chemotherapy (chemotherapy group) versus preoperative chemotherapy with postoperative chemoradiotherapy (chemoradiotherapy group) in patients with resectable gastric cancer. Patients with blood samples (plasma and white blood cells) available for at least two timepoitns during the course of treatment were included in this study. Potential self-selection bias or other biases were not identified.
The study was approved by the medical ethical committee of the Netherlands Cancer Institute and by the review boards of all participating centers. All patients provided oral and written informed consent.