Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers

Abstract

Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA.

Figure 1. Assay Schema.

(A) Blood is drawn before and after surgery. DNA from tumor is sequenced using the next-generation mate-pair sequencing (MPseq) protocol to identify chromosomal rearrangements. Several junctions are chosen to construct a personalized panel for each tumor. Percent of ctDNA out of total cfDNA is calculated at each time point of blood collection. (B) Number of junctions identified in cohort of 10 cases of serous stage 3 ovarian cancer. Count numbers are above the bars with the expected false negative count in parenthesis. cfDNA: cell-free DNA; ctDNA: circulating tumor DNA.

Figure 2. Next Generation mate-pair sequencing of primary Ovarian Cancer.

(A) Genome plot of exemplar case OC067. Chromosomes are listed on the left and right Y-axis’; basepair position is on the X axis. Grey cytobands indicate genomic loci bands. The height of the black dots each represents the average number of reads over 30 k bases. Magenta dots indicate small intrachromosomal rearrangements, while the magenta lines indicate interchromosomal rearrangements or larger intrachromosomal junctions. Green lines/dots indicate the junctions of interest monitored in the blood.

Figure 3. Detection of selected junctions in blood.

(A) Genome plot of case OC049, interpreted as in Fig. 2. (B) PCR validation of two selected junctions (as indicated) in tumor tissue of case OC049. (C,D) Agarose gels of qPCR showing junctions 1 and 2 of OC049 in pre-surgical plasma (C), pre-surgical serum, and post-surgical plasma (D).

Figure 4. Quantification of ctDNA fraction.

(A) Plot showing reference values for Ct (derived from qPCR) for housekeeping gene and junction of interest, calculated from a standard curve constructed using the Ct’s for four dilutions of DNA of corresponding tumor sample (B) Plot showing distribution of mate-pair reads corresponding to copy number variants in the tumor sample. Fraction of tumor was calculated using copy number probability density function. The largest peak corresponds to diploid copy number level (M_nat), the lower peak corresponds to a single copy deletion (M_del) and the third peak to gains. (C) Plot showing distribution of mate-pair reads as a function of coverage. The fraction of cells in the tumor harboring a specific junction was calculated using the expected coverage (M_cov), the tumor fraction (τ), and the number of mate-pairs associated with a junction (ν_j). (D) Count plot showing copy number changes (δ) at the locus of housekeeping gene ACTB with 2 examples showing wild-type 2 copy state (top) and copy loss (bottom).

Figure 5. Percent ctDNA.

Histogram showing the calculated percent (x-axis) of ctDNA in cfDNA for each detected rearrangement of each case. Error bars: calculated standard deviation.