Target selection for T-cell therapy in epithelial ovarian cancer: systematic prioritization of self-antigens

Adoptive T cell-receptor therapy (ACT) could represent a promising approach in the targeted treatment of epithelial ovarian cancer (EOC). However, the identification of suitable tumor-associated antigens (TAAs) as targets is challenging. We identified and prioritized TAAs for ACT and other immunotherapeutic interventions in EOC. A comprehensive list of pre-described TAAs was created and candidates were prioritized, using predefined weighted criteria. Highly ranked TAAs were immunohistochemically stained in a tissue microarray of 58 EOC samples to identify associations of TAA expression with grade, stage, response to platinum, and prognosis. Preselection based on expression data resulted in 38 TAAs, which were prioritized. Along with already published Cyclin A1, the TAAs KIF20A, CT45, and LY6K emerged as most promising targets, with high expression in EOC samples and several identified peptides in ligandome analysis. Expression of these TAAs showed prognostic relevance independent of molecular subtypes. By using a systematic vetting algorithm, we identified KIF20A, CT45, and LY6K to be promising candidates for immunotherapy in EOC. Results are supported by IHC and HLA-ligandome data. The described method might be helpful for the prioritization of TAAs in other tumor entities

A simple and robust real-time qPCR method for the detection of PIK3CA mutations

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status

Intra-operative myocardial ischaemia cannot be detected by analysis of transmitral inflow patterns in patients undergoing off-pump coronary surgery

SummaryBackground and objectiveTransmitral inflow patterns have been used for detection of myocardial ischaemia. However, its diagnostic value has not been tested in anaesthetized and mechanically ventilated patients undergoing coronary artery bypass graft surgery. METHODS: Transmitral inflow patterns were studied by transoesophageal Doppler echocardiography in 43 patients undergoing coronary artery bypass graft surgery without cardiopulmonary bypass after opening of the sternum (baseline) and during grafting of the left anterior descending artery. Peak early (E) and peak late (A) transmitral velocities and their ratio (E/A) were recorded. Myocardial ischaemia was defined by standard criteria using two-dimensional echocardiography and seven-lead electrocardiogram. RESULTS: Thirty-one patients (64 +/- 8 yr, 9 women) fulfilled the predefined inclusion criteria for analysis. During distal revascularization, 16 patients showed myocardial ischaemia and 15 did not. The use of vasoactive drugs, haemodynamic findings and transmitral inflow patterns were similar in both groups at baseline and during grafting. In the ischaemic group, E was 67.1 +/- 13.9 cm s-1 at baseline and 69.5 +/- 23.2 cm s-1 during grafting, and the E/A ratios were 1.3 +/- 0.3 and 1.4 +/- 0.9, respectively. In the non-ischaemic group, E was 64.0 +/- 17.1 cm s-1 at baseline and 60.9 +/- 14.8 cm s-1 during grafting, and the E/A ratios were 1.4 +/- 0.7 and 1.2 +/- 0.3, respectively. CONCLUSIONS: Analysis of Doppler findings of transmitral inflow patterns did not allow for detection of myocardial ischaemia during surgical revascularization of the myocardium

Axon guidance factor Slit2 inhibits neural invasion and metastasis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular and local invasion routes, which limit patient survival. In nerves and vessels, Slit2 and its Robo receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the Slit2-Robo system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent Slit2-Robo signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected Slit2 receptors Robo1 and Robo4 on epithelia, nerves and vessels in healthy pancreas and PDAC specimens, respectively. Slit2 mRNA expression was reduced in PDAC compared to non-transformed pancreatic tissues and cell lines, suggesting a reduction in Slit2-Robo pathway activity in PDAC. In support of this interpretation, restoring the Slit2 expression in Slit2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine Slit2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNAi-mediated silencing of Robo1 stimulated the motility of Slit2-competent PDAC cells. Furthermore, culture supernatants from Slit2-competent PDAC cells impaired migration of endothelial cells (HUVEC) whereas an N-terminal Slit2 cleavage fragment stimulated such migration. In vivo investigations of orthotopic pancreatic tumors with restored Slit2 expression demonstrated reduced invasion, metastasis and vascularization, with opposing effects produced by Robo1 silencing in tumor cells or sequestration of endogenous Slit2. Analysis of clinical specimens of PDAC showed that those with low Slit2-mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting Slit2-Robo signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion

Surgery with Radical Intent. Is There an Indication for G3 Neuroendocrine Neoplasms?

Background: While platinum-based chemotherapy represents the standard treatment for advanced grade 3 (G3) neuroendocrine neoplasms (NENs) according to the European Neuroendocrine Tumor Society guidelines, the role of radical-intended surgery in these patients, as well as the use of adjuvant chemotherapy, are still controversial. The aim of the present work is to describe, in a retrospective series of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) G3, the overall survival (OS) rate and risk factors for death after radical surgery. Secondary aims are the description of median recurrence-free survival (RFS) and of the role of adjuvant chemotherapy. Patients and Methods: Multicenter analysis of a series of stage I–III GEP-NEN G3 patients receiving radical surgery (R0/R1) with/without adjuvant chemotherapy was performed. Results: Sixty patients from eight neuroendocrine tumor (NET) referral centers, with median follow-up of 23 months (5–187 months) were evaluated. While 28.6% of cases had NET G3, 71.4% had neuroendocrine carcinoma G3 (NEC G3). The 2-year OS rate after radical surgery was 64.5%, with a statistically significant difference in terms of Ki67 threshold (cut-off 55%, P = 0.03) and tumor differentiation (NEC G3 vs. NET G3, P = 0.03). Median RFS after radical surgery was 14 months, and 2-year RFS rate was 44.9%. Use of adjuvant chemotherapy provided no benefit in terms of either OS or RFS in this series. Conclusions: Surgery with radical intent might represent a valid option for GEP-NEN G3 patients with locoregional disease, especially with Ki67 value ≤ 55%