Measurement of cyclin E genomic copy number and strand length in cell-free DNA distinguish malignant versus benign effusions.
OBJECTIVE
Previous studies have shown that the concentration of cell-free DNA was higher and its strand length longer in body fluids obtained from patients with cancer as compared to patients with benign diseases. We hypothesized that analysis of both DNA copy number and strand length of cell-free DNA from an amplified chromosomal region could improve the diagnosis of malignant diseases in body fluids.


METHODS
To test this hypothesis, we used ovarian cancer effusion as an example and applied a quantitative real-time PCR to measure the relative copy number and strand length of DNA fragments from one of the most frequently amplified genes, cyclin E, in ovarian serous carcinomas.


RESULTS
As compared with nonamplified chromosomal loci, including beta-actin, p53, 2p24.1, and 4p15.31, measurement of cyclin E DNA copy number (100 bp) had the best performance in distinguishing malignant (n = 88) from benign (n = 70) effusions after normalization to effusion volume or Line-1 DNA with areas under the receiver operating characteristics curve (AUC) of 0.832 and 0.847, respectively. Different DNA lengths of the cyclin E locus were further analyzed and we found that the AUC was highest by measuring the 400-bp cyclin E locus (AUC = 0.896). The AUC was improved to 0.936 when it was combined with the length integrity index as defined by the relative abundance of 400 bp cyclin E to 100 bp p53 loci. Cyclin E real-time PCR assay had a higher sensitivity (95.6%) than routine cytology examination (73.9%) and was able to diagnose false-negative cytology cases in this study.


CONCLUSIONS
The above findings indicate that measurement of the DNA copy number and strand length of the cyclin E locus is a useful cancer diagnostic tool.
Salani
Ritu
R
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
Davidson
Ben
B
Fiegl
Michael
M
Marth
Christian
C
Müller-Holzner
Elisabeth
E
Gastl
Guenther
G
Huang
Han-Yao
HY
Hsiao
Jui-Chi
JC
Lin
Her-Sheng
HS
Wang
Tian-Li
TL
Lin
Bai-Ling
BL
Shih
Ie-Ming
IeM
D019540:Area Under Curve; D002277:Carcinoma / Q000378:metabolism; D002474:Cell-Free System; D002874:Chromosome Mapping; D019927:Cyclin E / Q000096:biosynthesis* / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005188:False Negative Reactions; D016678:Genome; D006801:Humans; D008495:Medical Oncology / Q000379:methods; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D019927:Cyclin E; D017931:DNA Primers; D004247:DNA