Evaluation of DNA from the Papanicolaou test to detect ovarian and endometrial cancers

Abstract

Papanicolaou (Pap) smears have revolutionized the management of patients with cervical cancers by permitting the detection of early, surgically curable tumors and their precursors. In recent years, the traditional Pap smear has been replaced by a liquid-based method, which allows not only cytologic evaluation but also collection of DNA for detection of human papillomavirus, the causative agent of cervical cancer. We reasoned that this routinely collected DNA could be exploited to detect somatic mutations present in rare tumor cells that accumulate in the cervix once shed from endometrial or ovarian cancers. A panel of genes that are commonly mutated in endometrial and ovarian cancers was assembled with new whole-exome sequencing data from 22 endometrial cancers and previously published data on other tumor types. We used this panel to search for mutations in 24 endometrial and 22 ovarian cancers and identified mutations in all 46 samples. With a sensitive massively parallel sequencing method, we were able to identify the same mutations in the DNA from liquid Pap smear specimens in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). Prompted by these findings, we developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear specimen without previous knowledge of the tumor's genotype. When applied to 14 samples selected from the positive cases described above, the expected tumor-specific mutations were identified. These results demonstrate that DNA from most endometrial and a fraction of ovarian cancers can be detected in a standard liquid-based Pap smear specimen obtained during routine pelvic examination. Although improvements need to be made before applying this test in a routine clinical manner, it represents a promising step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies.

Fig. 1

Schematic demonstrating the principal steps of the procedure described in this study. Tumor cells shed from ovarian or endometrial cancers are carried into the endocervical canal. These cells can be captured by the brush used for performing a routine Pap smear. The brush contents are transferred into a liquid fixative, from which DNA is isolated. By means of massively parallel sequencing, this DNA is queried for mutations that indicate the presence of a malignancy in the female reproductive tract.

Fig. 2

Diagram of the modified Safe-SeqS assay used, which allowed for the simultaneous detection of mutations in 12 different genes. Top left: DNA templates from three exons of different genes (yellow, purple, and brown rectangles) to be queried for mutations. Note that only one of the templates contains a mutation (star) that exists before any sample preparatory steps or sequencing. Top right: Safe-SeqS primer pairs contain binding sites for universal primers (“UPS,” blue), a unique identifier (“UID,” red), and gene-specific sequences (colors match the targeted exon). Next, the templates and primers are combined into a single PCR compartment and a UID is attached to each targeted template, along with UPS binding sites, after a low number of PCR cycles (“UID assignment”). The Safe-SeqS primers are removed, and subsequent PCR is performed with primers containing UPS sites, as well as the sequences required for attachment to the sequencing instrument (“GP,” black) to prepare the templates for massively parallel sequencing. When mutations preexist in template DNA before sample preparation, all of the sequenced daughter molecules sharing the same UID will contain the same mutation (a “supermutant”). In contrast, artifactual mutations caused by sample preparation or sequencing are unlikely to be observed in most other daughter molecules sharing the same UID (“Artifact”). Note that only one of two DNA strands is depicted for clarity.

Fig. 3

Percent mutant alleles in liquid Pap smear specimens. The fraction of mutant alleles from each of 46 Pap specimens is depicted. The stage of each tumor is listed on the y axis. The x axis demonstrates the percent mutant allele fraction as determined by traditional Safe-SeqS. Mutant allele frequencies are higher than 10% in some cases but are depicted at 10% in this figure for clarity. Precise mutation frequencies are reported in table S3 for all samples.

Fig. 4

Heat map depicting the results of multiplex testing of 12 genes in Pap specimens. The PapGene test interrogates 46 gene regions, with each block on the y axis representing one region analyzed for the indicated gene. The 28 samples assessed (14 from control women without cancer and 14 from women with cancer) are indicated on the x axis. Mutations are indicated as colored blocks, with white indicating no mutation, yellow indicating a mutant fraction of 0.1 to 1%, orange indicating a mutant fraction of 1 to 10%, and red indicating a mutant fraction of >10%.