Researchers genetically analyzed DNA from cervical fluids obtained on routine Papanicolaou (Pap) testing to detect endometrial and ovarian cancers. The new test, called PapSEEK, incorporates assays for mutations in 18 genes as well as an assay to detect the presence of abnormal numbers of chromosomes in cells. Results of the study were published in Science Translational Medicine.
Prior research has shown that tumor DNA can be detected in the vaginal tract of women with ovarian cancer; in addition, endometrial and ovarian cancers shed cells that collect at the cervix, and tumor DNA can be found in the fluids obtained during routine Pap tests.
The study, led by Yuxuan Wang, from Johns Hopkins University School of Medicine in Baltimore, MD, used the DNA from Pap test fluid in a PCR-based, multiplex test to simultaneously assess genetic alterations that commonly occur in endometrial or ovarian cancers.
They also explored two ways to increase test sensitivity: by evaluating intrauterine samples closer to the anatomical sites of the tumors using a flexible, narrow brush covered by a retractable outer sheath, called a Tao brush, and by assessing mutations in both the plasma and Pap test fluid.
Samples were retrospectively collected from 1,658 individuals, including 656 patients with endometrial or ovarian cancers, and 1,002 healthy controls. All histopathology was re-reviewed by board-certified pathologists and data analysis was performed in a blinded manner.
In the Pap brush samples, the investigators used a sensitive, PCR-based error-reduction technology to identify mutations. Most DNA originates from normal cells, so the amount of DNA shed from neoplastic cells is typically a minor fraction of the total DNA.
Among 382 women with endometrial cancers, 81% had detectable mutations, including 78% of patients with early-stage disease (stages I and II) and 89% of patients with late-stage disease (stages III and IV). The most commonly mutated genes were consistent with previous genome-wide studies of endometrial cancers. The median mutant allele fraction (MAF) was 4.0%.
A total of 29% of 245 ovarian cancer patients had detectable mutations in their Pap brush samples. This included 28% of patients with early-stage disease and 30% of patients with late-stage disease. The most commonly mutated gene was consistent with previous genome-wide studies of this tumor type. The median MAF was 0.54%.
The assay was applied to 714 women without cancer and 1.3% had a detectable mutation, yielding a specificity of ~99%.
Tumor tissue was available from 83% and 84% of endometrial and ovarian cancer patients who donated Pap brush samples, respectively. Using the same multiplex assay applied to the Pap brush samples, a driver gene mutation could be identified in 98% of endometrial and 82% of ovarian cancer tissues.
In addition to somatic mutations, aneuploidy was detected in the Pap brush samples of 38% of the 382 patients with endometrial cancer and 11% of the 245 ovarian cancer patients. In contrast, of 714 women without cancer, only one tested positive on the aneuploidy assay for the Pap brush samples (specificity: ~100%).
A PapSEEK score was positive if it had either a mutation or an abnormal chromosome arm number. Of the Pap brush samples tested, positive PapSEEK scores were noted in 81% with endometrial cancers (78% and 92% with early- and late-stage disease, respectively), and 33% of women with ovarian cancers (34% and 33% with early- and late-stage disease, respectively).
Only 1.4% of the Pap brush samples from 714 women without cancer were PapSEEK-positive, yielding a specificity of ~99%.
A Tao brush allows direct sampling of the entire endometrial cavity without injury to the myometrium or contamination from the cervical canal. It was used to determine if more direct, minimally invasive sampling of the intrauterine cavity could increase the sensitivity for detecting gynecologic cancers.
PapSEEK detected genetic alterations in 93% of Tao brush samples collected from 123 patients with endometrial cancers, including 90% and 98% of patients with early- and late-stage disease, respectively. The most commonly mutated genes in the Tao brush samples were similar to those seen in the Pap brush samples. The median MAF was 24.7%, which was considerably higher than the 4% observed in the Pap brush samples.
Of the 51 women with ovarian cancers, genetic alterations detectable by PapSEEK were found in 45%, including 47% and 44% of patients with early- and late-stage cancers, respectively. Similarly, the most commonly mutated genes were consistent with that observed in the Pap brush samples. The median MAF was 0.88%, which was higher than the 0.54% noted in the Pap brush samples.
PapSEEK was applied to the Tao brush samples from 125 women without cancer. None of the women tested positive for mutations, yielding a specificity of 100%.
The authors hypothesized that ovarian cancers inaccessible by Pap or Tao brush sampling might be detectable by the presence of circulating tumor DNA (ctDNA) in plasma. Among women with ovarian cancer who donated both Pap brush and plasma samples, ctDNA was detected in the plasma of 43% of 83 patients. The sensitivity for ctDNA in plasma was higher in patients with late-stage tumors than in early-stage tumors (56% vs 35%, respectively).
In the Pap brush samples from the same cohort, 40% were positive by the PapSEEK test. Those with positive results only partially overlapped, resulting in 63% of patients who were positive for at least one of the two tests.
The authors acknowledge that the study has limitations. The patients in the retrospective cohort had cancer. In a screening setting, the cancers would be at an earlier stage, so the sensitivities for detection would be expected to be closer to the sensitivity for early-stage cancers observed.
“We designed and applied a multiplex PCR-based test (PapSEEK) to detect genetic alterations in Pap brush or Tao brush samples,” the authors wrote. “These samples are minimally invasive and conveniently obtained during routine office visits. Most endometrial cancers could be detected with PapSEEK: 93% with Tao brush and 81% with Pap brush. A substantial fraction of ovarian cancers could also be detected with PapSEEK: 45% with Tao brush and 33% with Pap brush.”
In addition, assays for ctDNA in plasma could be used in conjunction with PapSEEK on Pap brush samples, increasing the sensitivity of detecting ovarian cancer to 63%.
The investigators suggested that PapSEEK should be evaluated further in a large prospective study which includes patients at high risk for gynecologic cancers due to hereditary factors, obesity, or symptoms such as postmenopausal or dysfunctional uterine bleeding.
To read more about this study, click here.