Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing

Abstract

Clinical management of cancer patients could be improved through the development of noninvasive approaches for the detection of incipient, residual, and recurrent tumors. We describe an approach to directly identify tumor-derived chromosomal alterations through analysis of circulating cell-free DNA from cancer patients. Whole-genome analyses of DNA from the plasma of 10 colorectal and breast cancer patients and 10 healthy individuals with massively parallel sequencing identified, in all patients, structural alterations that were not present in plasma DNA from healthy subjects. Detected alterations comprised chromosomal copy number changes and rearrangements, including amplification of cancer driver genes such as ERBB2 and CDK6. The level of circulating tumor DNA in the cancer patients ranged from 1.4 to 47.9%. The sensitivity and specificity of this approach are dependent on the amount of sequence data obtained and are derived from the fact that most cancers harbor multiple chromosomal alterations, each of which is unlikely to be present in normal cells. Given that chromosomal abnormalities are present in nearly all human cancers, this approach represents a useful method for the noninvasive detection of human tumors that is not dependent on the availability of tumor biopsies.

Figures

Fig. 1

Schematic of analyses for direct detection of chromosomal alterations in plasma. The method uses next-generation paired-end sequencing of cell-free DNA isolated from plasma to identify chromosomal alterations characteristic of tumor DNA. Such alterations include copy number changes (gains and losses of chromosome arms) as well as rearrangements resulting from translocations, amplifications, or deletions.

Fig. 2

Copy number analysis of plasma samples. (A) The z scores for each chromosome arm indicate the number of SDs from the mean of the mapped read fraction of the plasma DNA from unaffected individuals (N1 to N10). Positive z scores indicate chromosome gains, whereas negative z scores indicate chromosome losses. Significant chromosome arm gains and losses were observed only in plasma samples from patients with cancer (CRC11 to CRC17 and BR1 to BR3). (B) The PA score was calculated as the number of SDs from the mean of the sum of the −log of the P values for the top five chromosome z scores of the 10 reference samples (N1 to N10). A PA score of 5.84 (horizontal line) was estimated to indicate aneuploidy in the plasma sample at a specificity greater than 99% (Student’s t distribution) (see Materials and Methods).

Fig. 3

Detection of tumor-specific rearrangements in plasma samples. The Circos plot at the top indicates the rearrangements identified in plasma samples from cancer patients (CRC11 to CRC17 and BR1 to BR3). The type and individual boundaries of the rearrangements are indicated in the lower table. No rearrangements were identified in plasma samples from unaffected individuals (N1 to N10). Rearrangements listed for sample CRC12 were identified in tumor DNA and confirmed in patient plasma, whereas those listed for all other samples were identified directly from patient plasma.

Fig. 4

Copy number analyses of tumor and serial plasma samples from patient CRC14. CRC14 primary tumor and plasma samples taken at various time points over 62 months of multimodality treatment were analyzed using DK in nonoverlapping 1-Mb windows and compared with unmatched normal plasma using the same methodology. The plasma samples were obtained at the time of initial evaluation (0 months), after extensive chemotherapy and surgical intervention (4 months), and at the time of cancer recurrence (62 months).

Fig. 5

Detection of circulating tumor DNA in breast and colon cancers using simulated copy number analyses. ROC analyses of simulated mixtures of breast cancer DNA (left) or colorectal cancer DNA (right) with normal plasma DNA using the PA score derived from the five chromosomal arm copy number alterations with the highest absolute z scores in each sample. Detection of 0.75% circulating tumor DNA could be achieved with a sensitivity of >90% and specificity of >99% using the equivalent of one HiSeq lane of sequencing and a fixed PA score threshold in both tumor types (see Materials and Methods). ROC analyses of a z score from a single chromosome arm, 17p, were similar to chance alone at this simulated tumor DNA concentration in the plasma.