Fig 1.
Study design and process pipeline for circulating tumor DNA (ctDNA) detection.
(A) Blood is drawn pre–and six weeks post–surgery in patients with endometrial cancer. DNA from the tumor is sequenced using the mate pair sequencing protocol. Junctions are detected and used to design individualized ctDNA qPCR assays to interrogate the blood for the presence of tumor DNA. (B) Primers are designed for selected junctions and undergo a pipeline to test for sufficient specificity and sensitivity to be used in the ctDNA assays.
Table 1.
Clinical characteristics of the endometrial cancer patients and circulating tumor DNA detection.
Fig 2.
Mate–Pair sequencing results from primary tumor DNA.
(A) Genome plot for primary tumor of case CTE024. 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 dots each represents the average number of reads over 30 k bases. Grey color indicates wild–type 2 copy state of DNA, blue indicates gains, red indicates losses. Black dots indicate small intrachromosomal rearrangements, while the black lines indicate interchromosomal rearrangements or larger intrachromosomal junctions. (B) Bar graph showing the number of chromosomal junctions detected in the primary tumor of 11 cases with a threshold of at least 3 supporting reads per junction. (C) Genome plot for cases CTE025, (D) CTE029, and (E) CTE031.
Fig 3.
Junction primer design and qPCR cfDNA detection results.
(A) Case CTE029. Left Panel: Junction plots of selected intrachromosomal junction of chromosome 3 with base positions 172MB–173MB (3a–3b). The middle line separates the 2 chromosomal areas involved in the junction. The lines across show the fragments that span the junction and support the rearrangement. The position of each read of a supporting fragment is located by a dot and color coded by strand, red for reads mapping to the reverse strand, blue for forward strand. The bridged coverage for the region is illustrated by the shaded area. The green dotted line on the y–axis indicates the bridged coverage averaged across the entire genome (normalized to estimate 2N and 1N). Genes within the region are displayed, indicating exon location and strand direction. General relative positions and directions of designed primers indicated. Middle panel: Linear plot of junction. Right Panel: Standard endpoint PCR of junction 3a–3b and amplification control product NAGK in pooled genomic DNA, patient tumor, and patient PBMCs. Sybr green qPCR amplicon melting curve for 3a–3b junction in patient 6–week post–surgical cfDNA. (B) Case CTE025. Left Panel: Junction plot of selected junction. Middle Panels: Sybr green qPCR melting curve of amplicons of 17a–17b junction in patient pre–and post–surgical cfDNA. Right Panel: qPCR melting curve for control product NAGK. (C) Case CTE031. Left Panel: Junction plot of selected junction. Middle Panels: Sybr green qPCR melting curve of amplicons of 5–17 junction in patient pre–and post–surgical cfDNA. Right Panel: qPCR melting curve for junction 5–17 in presurgical cfDNA following 30 cycles of pre–amplification.
Fig 4.
Heat map characterizing and correlating clinical findings to the presence or absence of detected circulating tumor DNA (ctDNA).
Abbreviations: CS, carcinosarcoma; D, detected; E, endometrioid; G, grade; LVSI, lymphovascular space invasion; Mx, Mixed; NA, not available; ND, not–detected; Neg, negative; P + PA LND, pelvic + para–aortic lymphadenectomy; Pos, Positive; S, Serous; UP LND, unilateral pelvic lymphadenectomy.