Characterisation of the immune-related transcriptome in resected biliary tract cancers

Although biliary tract cancers (BTCs) are known to have an inflammatory component, a detailed characterisation of immune-related transcripts has never been performed. In these studies, nCounter PanCancer Immune Profiling Panel was used to assess the expression of 770 immune-related transcripts in the tumour tissues (TTs) and matched adjacent tissues (ATs) of resected BTCs. Cox regression analysis and Kaplan-Meier methods were used to correlate findings with relapse-free survival (RFS). The first analysis in the TT and AT of an exploratory set (n = 22) showed deregulation of 39 transcripts associated with T-cell activation. Risk of recurrence was associated with a greater number of genes deregulated in AT in comparison to TT. Analysis in the whole set (n = 53) showed a correlation between AT cytotoxic T-lymphocyte antigen-4 (CTLA4) expression and RFS, which maintained statistical significance at multivariate analysis. CTLA4 expression correlated with forkhead box P3 (FOXP3) expression, suggesting enrichment in T regulatory cells. CTLA4 is known to act by binding to the cluster of differentiation 80 (CD80). No association was seen between AT CD80 expression and RFS. However, CD80 expression differentiated prognosis in patients who received adjuvant chemotherapy. We showed that the immunomodulatory transcriptome is deregulated in resected BTCs. Our study includes a small number of patients and does not enable to draw definitive conclusions; however, it provides useful insights into potential transcripts that may deserve further investigation in larger cohorts of patients. TRANSCRIPT PROFILING: Nanostring data have been submitted to GEO repository: GSE90698 and GSE906

Mismatch repair protein expression defects in endometrioid endometrial adenocarcinoma

Objectives: Endometrioid endometrial carcinoma (EEC) is the sentinel cancer in over half female patients with heritable mismatch repair (MMR) mutations as part of Lynch syndrome. Immunohistochemical testing for tumoural MMR-protein expression is the primary screening test identifying cases potentially harbouring familial cancer syndrome-related mutations and is also a predictive biomarker for immune-checkpoint blockade response.Methods: Following Data Protection and Ethical clearance by the University of Malta, 200 EEC cases were retrospectively identified and categorized into three arms: 151 cases above age 50 at diagnosis, 49 cases at or below age 50 at diagnosis and 30 controls with benign endometrial tissue sampling. H&E case slides were re-examined by an independent pathologist to confirm the diagnosis and identify the block best representing the tumour. Four new slides per case were recut and immunohistochemistry performed for MLH1, PMS2, MSH2, and MSH6 proteins. Protein expression was analysed semiquantitatively using Allred scoring.Results: 31% of the overall EEC cases were deficient for one or more MMR-proteins. Dual loss of the MLH1-PMS2 protein heterodimer was the most common deficiency, occurring in 24.5% of cases. Loss of MSH2-MSH6 protein expression represented 3.2% of MMR-deficient cases. Well differentiated tumours had a 76.5% proficiency rate as opposed to grade 2/3 disease with 53.2% and 52.9% proficiency rate respectively. There was no significant difference in MMR status when age 50 was used as a hypothetical testing threshold. After correcting for tumour grade, MLH1 and PMS2 expression was shown to be negatively correlated with age-at-diagnosis while MSH6 expression was positively correlated.Conclusion: Reflex MMR proficiency testing of all EEC cases is advisable, as using age 50 as a testing threshold would have missed 82.3% of MMR deficient cases. Prospective evidence is required to clarify the role semi-quantitative scoring plays in MMR status interpretation and patient management in the ever-evolving field of targeted therapeutics.peer-reviewe

Epithelioid trophoblastic tumour : a case with genetic linkage to a child born over seventeen years prior, successfully treated with surgery and pembrolizumab

Epithelioid trophoblastic tumours are rare neoplasms showing differentiation towards the chorion leave-type intermediate cytotrophoblast, with only a handful of cases being reported in the literature. These tumours are slow-growing and are typically confined to the uterus for extended periods of time. While the pathogenesis is unclear, they are thought to arise from a remnant intermediate trophoblast originating from prior normal pregnancies or, less frequently, gestational trophoblastic tumours. A protracted time period between the gestational event and tumour development is typical. This case describes a 49-year-old previously healthy female who presented with a completely asymptomatic uterine mass, discovered incidentally during a routine gynaecological assessment. The pathological analysis of the hysterectomy specimen confirmed an epithelioid trophoblastic tumour, involving the uterus and cervix. This is a rare gynaecological tumour. A comparative short tandem repeat analysis revealed genetic similarities to a previous healthy gestation seventeen years prior. She was successful treated with adjuvant pembrolizumab, with no evidence of disease recurrence to date.peer-reviewe

An overview of the role of long non-coding RNAs in human choriocarcinoma

Funding: The publication of this manuscript was funded by the European Cooperation in Science & Technology Program (EU COST)–COST Action CA18117: GYNOCARE- “European network for Gynaecological Rare Cancer research: From Concept to Cure”. R.D.F., S.S., A.F., S.A.O., J.J.O., M.P.W., J.B., M.S., P.O., A.Y., M.V.-S., B.E., A.G., and J.C.-A. are Action management committee members and/or form part of Working Groups 1–3, and J.C.-A. is the Chairperson of this Action.Choriocarcinoma (CC), a subtype of trophoblastic disease, is a rare and highly aggressive neoplasm. There are two main CC subtypes: gestational and non-gestational, (so called when it develops as a component of a germ cell tumor or is related to a somatic mutation of a poorly differentiated carcinoma), each with very diverse biological activity. A therapeutic approach is highly effective in patients with early-stage CC. The advanced stage of the disease also has a good prognosis with around 95% of patients cured following chemotherapy. However, advancements in diagnosis and treatment are always needed to improve outcomes for patients with CC. Long non-coding (lnc) RNAs are non-coding transcripts that are longer than 200 nucleotides. LncRNAs can act as oncogenes or tumor suppressor genes. Deregulation of their expression has a key role in tumor development, angiogenesis, differentiation, migration, apoptosis, and proliferation. Furthermore, detection of cancer-associated lncRNAs in body fluids, such as blood, saliva, and urine of cancer patients, is emerging as a novel method for cancer diagnosis. Although there is evidence for the potential role of lncRNAs in a number of cancers of the female genital tract, their role in CC is poorly understood. This review summarizes the current knowledge of lncRNAs in gestational CC and how this may be applied to future therapeutic strategies in the treatment of this rare cancer.publishersversionpublishe

Modulation of biliary cancer chemo-resistance through microRNA-mediated rewiring of the expansion of CD133+ cells

Changes in single microRNA (MIR) expression have been associated with chemo-resistance in Biliary Tract Cancer (BTC). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: high-throughput-screening of 997 LNA-MIR-inhibitors was performed in 6 CCA cell lines treated with Cisplatin-Gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with miRvana probes. MIR and gene expression was assessed by TaqMan-assay, RNA-sequencing and in-situ-hybridization in 4 indepedent cohorts of human BTC. Knock-out of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in-vitro and in-vivo. High-throughput-screening revealed that MIR1249-inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTC. In validation experiments, MIR1249-inhibition did not alter cell viability in untreated or DMSO-treated cells; however it did increase CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem niche of human BTC, as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion via the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of Cancer-Stem-Cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumour shrinkage after exposure to weekly CG, while WT models showed only stable disease over treatment

Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome

Longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the PROSPECT-C phase II colorectal cancer clinical trial

Sequential profiling of plasma cell-free DNA (cfDNA) holds immense promise for early detection of patient progression. However, how to exploit the predictive power of cfDNA as a liquid biopsy in the clinic remains unclear. RAS pathway aberrations can be tracked in cfDNA to monitor resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer. In this prospective phase II clinical trial of single-agent cetuximab in RAS wild-type patients, we combine genomic profiling of serial cfDNA and matched sequential tissue biopsies with imaging and mathematical modeling of cancer evolution. We show that a significant proportion of patients defined as RAS wild-type based on diagnostic tissue analysis harbor aberrations in the RAS pathway in pretreatment cfDNA and, in fact, do not benefit from EGFR inhibition. We demonstrate that primary and acquired resistance to cetuximab are often of polyclonal nature, and these dynamics can be observed in tissue and plasma. Furthermore, evolutionary modeling combined with frequent serial sampling of cfDNA allows prediction of the expected time to treatment failure in individual patients. This study demonstrates how integrating frequently sampled longitudinal liquid biopsies with a mathematical framework of tumor evolution allows individualized quantitative forecasting of progression, providing novel opportunities for adaptive personalized therapies