Center for Molecular Medicine Cologne

Ultrashort cell-free DNA fragments in blood identified: A potential new diagnostic target for precision oncology


Identification of a novel population of “ultrashort” cell-free DNA with G-quadruplex potential reduced abundance and G4-forming potential in blood plasma of healthy individuals than of patients with cancer.

Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA in the Journal Genome Research - Genome Res. 2021 Dec 20:gr.275691.121. DOI: 10.1101/gr.275691.121

DNA released in the blood is referred to as cell-free DNA (cfDNA). Since most cfDNA is derived from turnover of blood cells, the fragmentation profiles of healthy individuals reflect the chromatin structure of blood cells. In contrast, fragmentation profiles from individuals with cancer were distinct from that of healthy individuals. Their cfDNA is a mixture derived from the degraded genome of blood cells and cancer cells derived from cancer tissue.

Thus, analysis of cfDNA is promising for broad applications in clinical settings. Recent analyses have shown that cfDNAs are a group of molecules with high diversification suggesting a cfDNA fragmentation length of about 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. So far, the characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA remain unclear.

The current study describes the identification and discovery of a novel “ultra-short” population of cfDNA as a potential new diagnostic cancer biomarker performed in an international setting of researchers led by Dr. Irena Hudecova (Cancer Research UK Cambridge Institute), Dr. Chris Smith (Cancer Research UK Cambridge Institute), Dr. Robert Hänsel-Hertsch (Center for Molecular Medicine Cologne (CMMC) and CECAD Cologne, Medical Faculty University of Cologne and University Hospital Cologne), associate Prof. Dr. Florent Mouliere (Cancer Centre Amsterdam, Vrije Universiteit Amsterdam) and Dr. Nitzan Rosenfeld (Cancer Research UK Cambridge Institute).

The study redrawing the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments is published with the following title: Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA in the Journal Genome Research - Genome Res. 2021 Dec 20:gr.275691.121. DOI: 10.1101/gr.275691.121  - Online ahead of print.

In their study researchers developed an affinity bead-based protocol to efficiently sequence and analyze the source of the “ultrashort” cfDNA by short-read shallow whole genome sequencing (sWGS). Through the use of this novel workflow “magnetic bead-based DNA extraction and ssDNA-based library preparation (MB-ssDNA)”, the researchers identified and characterized a new population of plasma cfDNA molecules organized around ~50 bp. In cancer patients, these fragments included tumor-derived molecules at relative levels similar to those detected in DNA fragments >150 bp.  The researchers showed that “ultrashort” cfDNA fragments could be used to identify samples from cancer patients based on their (1) relative abundance in plasma, (2) DNA sequence context, (3) relationship with transcription start sites, and (4) abundance in regions associated with G-quadruplexes (G4s).

“The finding, that the ultrashort cfDNA largely originates from accessible chromatin regions of blood cells with an extraordinary potential to adopt four-stranded secondary structures - so called G-quadruplexes (G4) - is intriguing. These structures form in regions of DNA that are rich in one of its building blocks, guanine (G), when a single strand of the double-stranded DNA loops out and doubles back on itself, forming a four-stranded ‘knot’ in the genome. Our observations suggest an an association between “ultrashort” cfDNA fragments and regulatory mechanisms” Robert Hänsel-Hertsch comments, who led the bioinformatics analysis and is chairing the joint authorship of the study with Irena Hudecova and Christopher G. Smith.

In previous studies Robert Hänsel-Hertsch has shown that G-quadruplexes (1) play a role in transcription, a key step in reading the genetic code and creating proteins from DNA and (2) occur, in contrast to healthy cells and tissues, more frequently in the genome of human cancer cells and cancerous tissues. The current study has revealed the predominant existence of ultrashort cell-free DNA with a high potential to adopt G4 secondary structure in blood of healthy donors but not cancer patients.

“We are familiar with the concept that cfDNA originates from genomic regions where DNA is tightly wrapped around proteins to control DNA condensation. This study is the first time that we have found ultrashort cell-free DNA with an extraordinary G4-forming potential in the blood of healthy donors but substantially less in cancer patients. This finding was very surprising for all of us!”, Robert Hänsel-Hertsch comments. Since 2018 Hänsel-Hertsch - awardee of the CMMC Junior Research Group Program - is leading the Laboratory for Genome Biology at the CMMC.

The research was funded by Cancer Research UK, Center for Molecular Medicine Cologne, DFG CRC1399, CRUK Cambridge, ERC and the Dutch Cancer Fund.

Original Publication:
Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA
Irena Hudecova1,2,10 Christopher G. Smith1,2,10, Robert Hänsel-Hertsch1,3,4,5,10 Chandra Chilamakuri1, James A. Morris1,2, Aadhitthya Vijayaraghavan1,2, Katrin Heider1,2, Dineika Chandrananda1,2,9, Wendy N. Cooper1,2, Davina Gale1,2, Javier Garcia-Corbacho6, Simon Pacey2, Richard D. Baird2,7, Nitzan Rosenfeld1,2,11 and Florent Mouliere1,2,8,11
Journal: Genome Res. 2021 Dec 20:gr.275691.121. DOI: 10.1101/gr.275691.121  - Online ahead of print.

1Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge, UK
2Cancer Research UK Cambridge Centre, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK
3Center for Molecular Medicine Cologne (CMMC), Medical Faculty, University of Cologne and University Hospital, DE
4Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Univ. of Cologne and Univ. Hospital Cologne,
5Institute of Human Genetics, University Hospital Cologne, DE
6Clinical Trials Unit, Clinic Institute of Hematological and Oncological Diseases, Hospital Clinic, Barcelona, ES
7Dept. of Oncology, University of Cambridge, UK
8Dept. of Pathology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Centre Amsterdam, NL

Scientific Contact:
Dr . Robert Hänsel-Hertsch
Center for Molecular Medicine Cologne | Lab. for Genome Biology
•   CMMC JRG 10 - Robert Hänsel-Hertsch  and  Haensel-Hertsch Lab. for Genome Biolog
•   Instute for Human Genetics - associated member
•   CECAD, associated member