Low Impact of Clonal Hematopoiesis on the Determination of RAS Mutations by Cell-Free DNA Testing in Routine Clinical Diagnostics

Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics.

[1]  J. Lindberg,et al.  ESMO recommendations on the use of circulating tumour DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group. , 2022, Annals of oncology : official journal of the European Society for Medical Oncology.

[2]  J. Reis-Filho,et al.  Delivering precision oncology to patients with cancer , 2022, Nature Medicine.

[3]  N. Kraut,et al.  Expanding the Reach of Precision Oncology by Drugging All KRAS Mutants , 2022, Cancer discovery.

[4]  Myung Ah Lee,et al.  Sotorasib for previously treated colorectal cancers with KRASG12C mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial. , 2021, The Lancet. Oncology.

[5]  A. Biankin,et al.  Cancer Biomarkers in the era of precision oncology: Addressing the needs of patients and health systems. , 2021, Seminars in cancer biology.

[6]  P. Jänne,et al.  Abstract LB002: Mechanisms of acquired resistance to KRAS G12C inhibition in cancer , 2021, Experimental and Molecular Therapeutics.

[7]  V. Fedele,et al.  Targeting KRAS: The Elephant in the Room of Epithelial Cancers , 2021, Frontiers in Oncology.

[8]  A. Chakraborty KRASG12C inhibitor: combing for combination. , 2020, Biochemical Society transactions.

[9]  R. Yamashita,et al.  Clinical utility of circulating tumor DNA sequencing in advanced gastrointestinal cancer: SCRUM-Japan GI-SCREEN and GOZILA studies , 2020, Nature Medicine.

[10]  P. Ascierto,et al.  Circulating Tumor DNA Testing Opens New Perspectives in Melanoma Management , 2020, Cancers.

[11]  D. Steensma,et al.  Implications of Clonal Hematopoiesis for Precision Oncology. , 2020, JCO precision oncology.

[12]  Hiu Ting Chan,et al.  Clinical significance of clonal hematopoiesis in the interpretation of blood liquid biopsy , 2020, Molecular oncology.

[13]  S. Mortimer,et al.  Spectrum of driver mutations and clinical impact of circulating tumor DNA analysis in non–small cell lung cancer: Analysis of over 8000 cases , 2020, Cancer.

[14]  T. Druley,et al.  The evolutionary dynamics and fitness landscape of clonal hematopoiesis , 2020, Science.

[15]  C. Curtis Quantifying mutations in healthy blood , 2020, Science.

[16]  N. Normanno,et al.  Targeted sequencing analysis of cell-free DNA from metastatic non-small-cell lung cancer patients: clinical and biological implications , 2020, Translational lung cancer research.

[17]  N. Chen,et al.  Discovery of a covalent inhibitor of KRASG12C (AMG 510) for the treatment of solid tumors. , 2019, Journal of medicinal chemistry.

[18]  David R. Jones,et al.  High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants , 2019, Nature Medicine.

[19]  N. Rosenfeld,et al.  Real-World Utility of an Amplicon-Based Next-Generation Sequencing Liquid Biopsy for Broad Molecular Profiling in Patients With Advanced Non–Small-Cell Lung Cancer , 2019, JCO precision oncology.

[20]  G. Botti,et al.  The Presence of Concomitant Mutations Affects the Activity of EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small Cell Lung Cancer (NSCLC) Patients , 2019, Cancers.

[21]  M. Ye,et al.  Biological background of the genomic variations of cf-DNA in healthy individuals , 2019, Annals of oncology : official journal of the European Society for Medical Oncology.

[22]  N. Normanno,et al.  The role of circulating free DNA in the management of NSCLC , 2018, Expert review of anticancer therapy.

[23]  Sharyn I. Katz,et al.  Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non–Small Cell Lung Cancer , 2019, JAMA oncology.

[24]  M. Speicher,et al.  Current and future perspectives of liquid biopsies in genomics-driven oncology , 2018, Nature Reviews Genetics.

[25]  N. Normanno,et al.  The liquid biopsy in the management of colorectal cancer patients: Current applications and future scenarios. , 2018, Cancer treatment reviews.

[26]  B. Chabner,et al.  Application of Cell-free DNA Analysis to Cancer Treatment. , 2018, The New England journal of medicine.

[27]  C. Paweletz,et al.  False-Positive Plasma Genotyping Due to Clonal Hematopoiesis , 2018, Clinical Cancer Research.

[28]  S. Ikeda,et al.  Genomic Alterations in Circulating Tumor DNA from Diverse Cancer Patients Identified by Next-Generation Sequencing. , 2017, Cancer research.

[29]  Jacqueline A. Hall,et al.  Review of the implementation of plasma ctDNA testing on behalf of IQN Path ASBL: a perspective from an EQA providers’ survey , 2017, Virchows Archiv.

[30]  James D. Brenton,et al.  Liquid biopsies come of age: towards implementation of circulating tumour DNA , 2017, Nature Reviews Cancer.

[31]  T. Druley,et al.  Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults , 2016, Nature Communications.

[32]  G. Botti,et al.  Limits and potential of targeted sequencing analysis of liquid biopsy in patients with lung and colon carcinoma , 2016, Oncotarget.