Giampero (John) A. Martignetti, MD, PhD
A targeted liquid biopsy may have the potential to serve as an early diagnostic tool for endometrial cancer, according to a case study published in The Cold Spring Harbor Journal of Molecular Case Studies
The study identified tumor-specific mutations in a patient with no clinical or pathologic indication of cancer approximately 1 year prior to her diagnosis, suggesting that analysis of a patient’s uterine lavage fluid can be used to screen for cancer-specific mutations prior to diagnosis.
“Taken together, we believe all these results establish both proof of principle for cancer screening by liquid biopsy and the paradigm for collection and analysis of relevantly targeted samples beyond blood,” wrote the authors, led by John A. Martignetti, MD, PhD, Network Director of the Laboratory for Translational Research at the Western Connecticut Health Network.
The targeted liquid biopsy technique was tested in a 67-year old female with a BMI of >34 and no family history of breast, pancreatic, ovarian, or endometrial cancer. The patient received a hysteroscopy, after which 10 mL of lavage fluid was collected and analyzed against a 12-gene endometrial cancer typing panel. The 12 genes that were accounted for included PTEN, PIK3CA, TP53, CTNNB1, KRAS, FGFR2, FBXW7, RB1, ATM, APC, ARID1A
, and PIK3R1
Molecular analysis of the lavage cell pellet revealed 5 driver mutations with varying allele frequencies and sequencing coverages: PTEN
R130G (1.3%/107 reads) and S338fs
(1.1%/349 reads); PIK3CA
N345I (4.9%/379 reads); FBXW7
R505C (5.0%/467 reads); and FGFR2
S252W (5.1%/355 reads).
Comparison against The Cancer Genome Atlas (TCGA) data set for endometrial cancer revealed an overlap between all of the identified genes and those linked to endometrial cancer. Most notably was PTEN
R130, which is found in approximately 48% of patients with endometrial cancer.
Ten months later, the patient reported postmenopausal bleeding. Subsequent pathology revealed a single lesion of endometrioid adenocarcinoma. Molecular analysis of the tumor through laser-capture microdissection (LCM) revealed 2 of the same somatic mutations––PTEN
R130G and Ser338fs
––that had been identified in the uterine lavage cell pellet, but with greater allele frequencies (>10%).
To account for the possibility that the mutations occurred by chance, researchers assumed that every position within the 12-gene sequencing panel had the same probability of mutating. The likelihood that the mutations in both the lavage and LCM presented by chance was extremely low (P
< 3 x 10-7).
“[By] using an integrated liquid biopsy-based and cancer-targeted sampling approach, we have established a benchmark for earliest endometrial cancer detection,” reported Martignetti et al.
Although the mutations that were identified carried a higher likelihood of developing endometrial cancer, there was no way of proving the timing and likelihood of onset. In the original prospective study in which the patient had enrolled, sequencing cellular and cell-free DNA (cfDNA) isolated from uterine lavage fluid detected 100% of existing endometrial cancers.2
Pulling from that study, researchers tested patients for the same PTEN
mutations that had been identified in the primary patient. Of the more than 100 patients on trial, 5 patients harbored the PTEN
The eldest of the 5 women was 67 years old with a BMI >38 and had been brought in as a result of postmenopausal bleeding. At the time of hysteroscopy, high-grade serous adenocarcinoma and grade 3 endometrioid adenocarcinoma had already been detected. Ten driver mutations were found in her lavage fluid, including a PTEN
R130Q mutation in the cell pellet and cfDNA at allele frequencies of 22% and 39%, respectively.
The other 4 women were significantly younger (range, 37-51) with BMIs that ranged from 19 to 34. Three women had been evaluated for abnormal uterine bleeding and the fourth for dysmenorrhea; 2 patients also had endometriosis. However, these patients showed no evidence of precancerous lesions or cancer at the time of hysteroscopy and lavage nor during the more than 2-year follow-up.
Though the case study sets a precedent for early detection, more research is needed to understand the relationship between somatic mutations and the potential development to preneoplastic lesions and cancer proliferation.
“Moving beyond the previously successful goal of TCGA in cataloging the genomic landscape of advanced disease, the future goal would be to integrate the multiomics landscape and order the sequence of events and immunologic features that result in cancer. By way of a marathon analogy, interviewing only the elite runners as they cross the finish line reveals nothing about the larger total field of runners, those who dropped out and why, and being able to reasonably predict whether the remaining runners will complete or abandon the course [as detailed in a Pre-Cancer Genome Atlas],” concluded Martignetti et al.
- Martignetti J, Pandya D, Nagarsheth N, et al. Detection of endometrial precancer by a targeted gynecologic cancer liquid biopsy [published online October 9, 2018]. Cold Spring Harb Mol Case Stud. doi: 10.1101/mcs.a003269.
- Nair N, Camacho-Vanegas O, Rykunov D, et al. Genomic analysis of uterine lavage fluid detects early endometrial cancers and reveals a prevalent landscape of driver mutations in women without histopathologic evidence of cancer: a prospective cross-sectional study. [published online December 27, 2016]. PLOS Med. doi.org/10.1371/journal.pmed.1002206.