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File:PapSEEK workflow.png
PapSEEK workflow

PapSEEK is an experimental screening test for detecting some endometrial and ovarian cancers.[1]

It is based on the sequencing and genetic analyses of DNA obtained from a routine Pap test.[2] Using a multiplex PCR-based test, PapSEEK detects somatic mutations in the 18 most commonly mutated genes in endometrial and ovarian cancers.[2] Assays are also used to detect aneuploidy in samples that may not have mutations in any of the 18 genes.[2]

PapSEEK aims to increase sensitivity of cancer screening in two ways: 1. Using a “Tao brush” that allows for collection of samples that are anatomically closer to the sites of the tumor.[2] 2. Combine Pap test fluid sample analysis with plasma analysis results.[2] The outcome of PapSEEK demonstrates an increased detection of early stage gynecological cancers using mutation-based diagnostics.[2]


The Pap test is highly valuable for early detection of cervical cancers through screening of populations and reducing the mortality rate of people with cervical cancer. However, the Pap test can only detect cervical cancer and not other gynecological cancers such as endometrial and ovarian cancers.[3][4][5] Due to this discrepancy, the most lethal gynecological cancers in countries where Pap test is routinely performed are endometrial and ovarian cancers[6] Together, these two cancers are the third-leading cause of cancer mortality in women and account for approximately 25,000 deaths each year in the United States.[6]

Ovarian cancer is even more difficult to detect and often diagnosed at a later stage, leading to a high mortality rate.[6] Screening the general population for ovarian cancers with the current diagnostic approaches, including TVUS, results in unnecessary invasive interventions that are not recommended.[2] One of the most common ovarian cancers with the lowest survival rates is high-grade serous carcinoma (HGSC).[2] Recently, more evidence supports the fact that HGSC is initiated in the fallopian tube and migrates to the ovarian surface for implantation.[7][8][9][10] Moreover, a recent study suggests that most early-stage HGSCs originate in extraovarian sites.[11]


A regular Papanicolaou (Pap) test is first performed to collect cells from the cervical opening (cervical os).[12] DNA recovered is then amplified to detect for mutations in 18 genes as well as aneuploidy[12].[13][14]

Detection of somatic mutations

A multiplex PCR-based approach is used to detect mutations commonly found in endometrial and ovarian cancers.[2] 139 primers were designed to amplify distinct regions within the 18 genes of interest: AKT1, APC, BRAF, CDKN2A, CTNNB1, EGFR, FBXW7, FGFR2, KRAS, MAPK1, NRAS, PIK3CA, PIK3R1, POLE, PPP2R1A, PTEN, RNF43, and TP53.[12][13][14] Each amplification segment is about 110- to 142-bp in length, covering a total of 9392 nucleotides in the DNA.[2] Three multiplex PCRs with non-overlapping amplicons were performed for each sample.[2]

To capture the minor fraction of neoplasm DNA within the Pap smear sample, a PCR-based error-reduction technology ‘Safe-Sequencing System (Safe-SeqS)’ is used to amplify the DNA.[15] One primer from each primer pair contains a 14-nt unique molecular identifier (UMI).[2] Hence, amplified DNA segments containing the same UMI are seen as segments originating from a common template. In this way, rare mutations can be amplified and detected without being affected by PCR-bias.

Detection of aneuploidy

In addition to somatic mutation analysis, PapSEEK also evaluates sample aneuploidy to spot cancers with no mutation in any of the 18 genes assessed.[2] An amplicon-based approach is employed to detect aneuploidy.[16] In each sample, a single primer pair is used to amplify ~38,000 loci of long interspersed nucleotide elements (LINES) throughout the genome.[16] LINEs are dispersed widely on all chromosomes, making it a great primer binding sequence to detect gains and losses of chromosome arms. Amplified segments are then sequenced by the Illumina platform.[2] Data obtained are processed using the Within-Sample AneupLoidy DetectiOn (WALDO) software.[17] WALDO uses supervised machine learning to categorize aneuploid and euploid samples.[17] Samples having a support vector machine (SVM) score above a given threshold are classified as aneuploid. Samples having a gain of chromosome arm 7q and 8q, which are often gained in ovarian and endometrial cancers,[18][19] are also recognized as aneuploid by the software.[2]

Combining the detection for somatic mutations and aneuploidy, PapSEEK screens Pap brush and Tao brush samples for endometrial and ovarian cancers. A sample is reported positive if it contains either a mutation or a gain or loss in chromosome arm.[2]

Tao brush vs Pap brush

File:Labeled pap bush uterine.png
Pap brush sampling technique
File:Labeled tao brush.png
Tao brush sampling

One possible way to improve PapSEEK's sensitivity is to collect cells from the intrauterine cavity (compared to the endocervical canal in regular Pap test).[2] Tao brush IUMC Endometrial Sampler (Tao brush), rather than Pap brush, is used for this purpose.[20] Tao brush is a Food and Drug Administration (FDA)-approved tool for endometrial sampling without the need for anesthesia.[2] The brush is equipped with a retractable outer sheath that cover the brush completely during insertion and removal, preventing injury to the myometrium and contamination from endocervix and vagina.[20]

During intrauterine sampling, Tao brush is gently inserted to the level of uterine fundus. The outer sheath is then pulled back to allow direct contact of the brush with the endometrium. Then, the brush is rotated 360° clockwise and then counterclockwise to collect tissue sample. The sheath is then replaced, and the device is removed.[20] DNA recovered from tissues collected is then analysed for somatic mutations and aneuploidy.

PapSEEK in plasma

Another way to improve PapSEEK's sensitivity is to assess mutations in both Pap test sample and plasma sample. Tumor cells shed free DNA called "circulating tumor DNA (ctDNA)" into peripheral blood.[21] Collecting plasma samples can prevent contamination from cells, and thus plasma has been proven to be a superior source of ctDNA.[22]


  1. "Experimental Test Detects Endometrial and Ovarian Cancers" (in en). 2018-05-02. 
  2. 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 Wang, Yuxuan; Li, Lu; Douville, Christopher; Cohen, Joshua D.; Yen, Ting-Tai; Kinde, Isaac; Sundfelt, Karin; Kjær, Susanne K. et al. (2018-03-21). "Evaluation of liquid from the Papanicolaou test and other liquid biopsies for the detection of endometrial and ovarian cancers". Science Translational Medicine 10 (433): eaap8793. doi:10.1126/scitranslmed.aap8793. ISSN 1946-6242. PMC 6320220. PMID 29563323. 
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  5. Zhao, Chengquan; Florea, Anca; Onisko, Agnieszka; Austin, R. Marshall (September 2009). "Histologic follow-up results in 662 patients with Pap test findings of atypical glandular cells: Results from a large academic womens hospital laboratory employing sensitive screening methods". Gynecologic Oncology 114 (3): 383–389. doi:10.1016/j.ygyno.2009.05.019. ISSN 0090-8258. PMID 19501894. 
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  11. Morency, Elizabeth; Leitao, Mario M.; Soslow, Robert A. (May 2016). "Low-stage high-grade serous ovarian carcinomas: support for an extra-ovarian origin". International Journal of Gynecological Pathology : Official Journal of the International Society of Gynecological Pathologists 35 (3): 222–229. doi:10.1097/PGP.0000000000000256. ISSN 0277-1691. PMC 5533190. PMID 26630225. 
  12. 12.0 12.1 12.2 Kinde, I.; Bettegowda, C.; Wang, Y.; Wu, J.; Agrawal, N.; Shih, I.-M.; Kurman, R.; Dao, F. et al. (2013-01-09). "Evaluation of DNA from the Papanicolaou Test to Detect Ovarian and Endometrial Cancers". Science Translational Medicine 5 (167): 167ra4. doi:10.1126/scitranslmed.3004952. ISSN 1946-6234. PMC 3757513. PMID 23303603. 
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  21. Siravegna, Giulia; Marsoni, Silvia; Siena, Salvatore; Bardelli, Alberto (2017-03-02). "Integrating liquid biopsies into the management of cancer". Nature Reviews Clinical Oncology 14 (9): 531–548. doi:10.1038/nrclinonc.2017.14. ISSN 1759-4774. PMID 28252003. 
  22. Nikolaev, Sergey; Lemmens, Laure; Koessler, Thibaud; Blouin, Jean-Louis; Nouspikel, Thierry (February 2018). "Circulating tumoral DNA: Preanalytical validation and quality control in a diagnostic laboratory". Analytical Biochemistry 542: 34–39. doi:10.1016/j.ab.2017.11.004. ISSN 0003-2697. PMID 29137972.