Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression.

Analyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors' evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.

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.

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network.

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.

EV-associated miRNAs from pleural lavage as potential diagnostic biomarkers in lung cancer.

Lung cancer is the leading cause of cancer-related deaths among men and women in the world, accounting for the 25% of cancer mortality. Early diagnosis is an unmet clinical issue. In this work, we focused to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the pleural lavage, a proximal fluid in lung cancer patients, as a source of potential biomarkers. We isolated EVs by ultracentrifuge method from 25 control pleural fluids and 21 pleural lavages from lung cancer patients. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 288 out of the 754 miRNAs that were contained in the OpenArray. The differential expression analysis yielded a list of 14 miRNAs that were significantly dysregulated (adj. p-value < 0.05 and logFC lower or higher than 3). Using Machine Learning approach we discovered the lung cancer diagnostic biomarkers; miRNA-1-3p, miRNA-144-5p and miRNA-150-5p were found to be the best by accuracy. Accordance with our finding, these miRNAs have been related to cancer processes in previous studies. This results opens the avenue to the use of EV-associated miRNA of pleural fluids and lavages as an untapped source of biomarkers, and specifically, identifies miRNA-1-3p, miRNA-144-5p and miRNA 150-5p as promising biomarkers of lung cancer diagnosis.

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.

EV-associated miRNAs from peritoneal lavage as potential diagnostic biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Current systematic methods for diagnosing have inherent limitations so development of a minimally-invasive diagnosis, based on the identification of sensitive biomarkers in liquid biopsies could therefore facilitate screening among population at risk. METHODS: In this study, we aim to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the peritoneal lavage as a source of potential miRNA diagnostic biomarkers. We isolated EVs by ultracentrifugation from 25 ascitic fluids and 25 peritoneal lavages from non-cancer and CRC patients, respectively. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 371 miRNAs. RESULTS: 210 miRNAs were significantly dysregulated (adjusted p value < 0.05 and abs(logFC) ≥ 1). The top-10 miRNAs, which had the AUC value higher than 0.95, were miRNA-199b-5p, miRNA-150-5p, miRNA-29c-5p, miRNA-218-5p, miRNA-99a-3p, miRNA-383-5p, miRNA-199a-3p, miRNA-193a-5p, miRNA-10b-5p and miRNA-181c-5p. CONCLUSIONS: This finding opens the avenue to the use of EV-associated miRNA of peritoneal lavages as an untapped source of biomarkers for CRC.

Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis.

BACKGROUND: Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary. METHODS: We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling. FINDINGS: The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5-99·7), 97·7% sensitivity (96·1-99·2), 88·6% positive predictive value (85·8-91·3), and 99·9% negative predictive value (99·9-100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42-7·38]; log-rank p=0·0029). INTERPRETATION: We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients. FUNDING: European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.

Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies.

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2(-16)), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

Translokin (Cep57) interacts with cyclin D1 and prevents its nuclear accumulation in quiescent fibroblasts.

Nuclear accumulation of cyclin D1 because of altered trafficking or degradation is thought to contribute directly to neoplastic transformation and growth. Mechanisms of cyclin D1 localization in S phase have been studied in detail, but its control during exit from the cell cycle and quiescence is poorly understood. Here we report that translokin (Tlk), a microtubule-associated protein also termed Cep57, interacts with cyclin D1 and controls its nucleocytoplasmic distribution in quiescent cells. Tlk binds to regions of cyclin D1 also involved in binding to cyclin-dependent kinase 4 (Cdk4), and a fraction of cyclin D1 associates to the juxtanuclear Tlk network in the cell. Downregulation of Tlk levels results in undue nuclear accumulation of cyclin D1 and increased Cdk4-dependent phosphorylation of pRB under quiescence conditions. In turn, overexpression of Tlk prevents proper cyclin D1 accumulation in the nucleus of proliferating cells in an interaction-dependent manner, inhibits Cdk4-dependent phosphorylation of pRB and hinders cell cycle progression to S phase. We propose that the Tlk acts as a key negative regulator in the pathway that drives nuclear import of cyclin D1, thus contributing to prevent pRB inactivation and to maintain cellular quiescence.