Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

EV-Associated miRNAs from Peritoneal Lavage are a Source of Biomarkers in Endometrial Cancer.

Endometrial cancer (EC) is the sixth most common cancer in women worldwide and is responsible for more than 89,000 deaths every year. Mortality is associated with presence of poor prognostic factors at diagnosis, i.e., diagnosis at an advanced stage, with a high grade and/or an aggressive histology. Development of novel approaches that would permit us to improve the clinical management of EC patients is an unmet need. In this study, we investigate a novel approach to identify highly sensitive and specific biomarkers of EC using extracellular vesicles (EVs) isolated from the peritoneal lavage of EC patients. EVs of peritoneal lavages of 25 EC patients were isolated and their miRNA content was compared with miRNAs of EVs isolated from the ascitic fluid of 25 control patients. Expression of the EV-associated miRNAs was measured using the Taqman OpenArray technology that allowed us to detect 371 miRNAs. The analysis showed that 114 miRNAs were significantly dysregulated in EC patients, among which eight miRNAs, miRNA-383-5p, miRNA-10b-5p, miRNA-34c-3p, miRNA-449b-5p, miRNA-34c-5p, miRNA-200b-3p, miRNA-2110, and miRNA-34b-3p, demonstrated a classification performance at area under the receiver operating characteristic curve (AUC) values above 0.9. This finding opens an avenue for the use of EV-associated miRNAs of peritoneal lavages as an untapped source of biomarkers for EC.

Genetic analysis of uterine aspirates improves the diagnostic value and captures the intra-tumor heterogeneity of endometrial cancers.

Endometrial cancer is the most common cancer of the female genital tract in developed countries. Although the majority of endometrial cancers are diagnosed at early stages and the 5-year overall survival is around 80%, early detection of these tumors is crucial to improve the survival of patients given that the advanced tumors are associated with a poor outcome. Furthermore, correct assessment of the pre-clinical diagnosis is decisive to guide the surgical treatment and management of the patient. In this sense, the potential of targeted genetic sequencing of uterine aspirates has been assessed as a pre-operative tool to obtain reliable information regarding the mutational profile of a given tumor, even in samples that are not histologically classifiable. A total of 83 paired samples were sequenced (uterine aspirates and hysterectomy specimens), including 62 endometrioid and non-endometrioid tumors, 10 cases of atypical hyperplasia and 11 non-cancerous endometrial disorders. Even though diagnosing endometrial cancer based exclusively on genetic alterations is currently unfeasible, mutations were mainly found in uterine aspirates from malignant disorders, suggesting its potential in the near future for supporting the standard histologic diagnosis. Moreover, this approach provides the first evidence of the high intra-tumor genetic heterogeneity associated with endometrial cancer, evident when multiple regions of tumors are analyzed from an individual hysterectomy. Notably, the genetic analysis of uterine aspirates captures this heterogeneity, solving the potential problem of incomplete genetic characterization when a single tumor biopsy is analyzed.

Modeling glands with PTEN deficient cells and microscopic methods for assessing PTEN loss: endometrial cancer as a model.

PTEN is an important tumor suppressor gene. Interpreting PTEN deficiency in the appropriate microscopic context of cancer may be important to understand its role in tumor development and progression. This may be particularly relevant in heterogeneous tumors. Here, we discuss the usefulness of 3D cultures in understanding the consequences of PTEN inactivation in tissue architecture. Afterwards, we discuss the role of immunohistochemistry and fluorescent in situ hybridization in assessing PTEN loss in tumors. In this review, endometrial carcinoma is used as a model.

Gene expression microarray-based assay to determine tumor site of origin in a series of metastatic tumors to the ovary and peritoneal carcinomatosis of suspected gynecologic origin.

The origin of the primary tumor is sometimes difficult to determine in peritoneal and ovarian metastases. A series of 25 metastatic tumors to the ovary and 7 cases of peritoneal carcinomatosis of suspected gynecologic origin were collected. Total RNA was extracted from frozen tumor tissue and studied by the Tissue of Origin-Frozen test, a microarray-based gene expression test from Pathwork Diagnostics (Redwood City, CA). Independently, formalin-fixed, paraffin-embedded tumor tissue was subjected to pathologic analysis. Immunohistochemical stains included keratins 7 and 20, estrogen and progesterone receptors, CDX2, villin, CEA, WT-1, TTF-1, mammoglobin, GCDF-15, and CD31. Clinical data were considered as gold standard, and after clinicopathologic evaluation, the tissue of origin was found in 29 cases. The Tissue of Origin-Frozen test correctly identified the ovary as site of origin in 7 of 7 peritoneal carcinomatosis cases, whereas immunohistochemical stains only allowed appropriate recognition in 5. In addition, the Tissue of Origin-Frozen test identified correctly the site of origin in 18 of the 22 metastatic tumors to the ovary with known origin. In the remaining 4 tumors, the correct origin was the second option in 2 cases and was not determined in the other 2. Immunohistochemistry correctly identified the site of origin in 17 of these 22 ovarian metastases. A combination of Tissue of Origin-Frozen and immunohistochemistry correctly identified the site of origin in 19 of 22 ovarian metastases of known origin. Although conventional pathologic examination and immunohistochemistry are commonly used for assessing the tumor site of origin, Tissue of Origin testing can be useful in difficult cases.