Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Magnetic Resonance Imaging Identified Brain Ischaemia in Symptomatic Patients Undergoing Carotid Endarterectomy Is Related to Histologically Apparent Intraplaque Haemorrhage

Objective: Intraplaque haemorrhage (IPH) has been independently associated with a higher risk of future ipsilateral stroke in patients with carotid artery stenosis. Evaluation of plaque characteristics may contribute to risk assessment of recurrent (silent) cerebrovascular events in order to prioritise patients for timing of treatment. It is unknown if patients showing histologically apparent IPH also have increased risk of silent ischaemic brain lesions in the waiting period between index event and revascularisation. Methods: A retrospective analysis was performed based on prospectively collected data of patients included simultaneously in the magnetic resonance imaging (MRI) substudy of the International Carotid Stenting Study and Athero-Express biobank. Patients randomised for carotid endarterectomy (CEA) underwent surgery between 2003 and 2008. Brain MRI was performed one to seven days prior to CEA. Plaques were histologically examined for presence of IPH. The primary outcome parameter was presence of silent ipsilateral brain ischaemia on magnetic resonance diffusion weighted imaging (MR-DWI) appearing hypo or isointense on apparent diffusion coefficient. Results: Fifty-three patients with symptomatic carotid stenosis meeting the study criteria were identified, of which 13 showed one or more recent ipsilateral DWI lesion on pre-operative scan. The median time between latest ipsilateral neurological event and revascularisation was 45 days (range 6–200) in DWI negative patients vs. 34 days (range 6–74, p = .16) in DWI positive patients. IPH was present in 24/40 (60.0%) DWI negative patients vs. 12/13 (92.3%) DWI positive patients (OR 8.00; 95% CI 0.95–67.7, p = .06). Multivariable logistic regression analysis correcting for age and type of index event revealed that IPH was independently associated with DWI lesions in the waiting period till surgery (OR 10.8; 95% CI 1.17–99.9, p = .04). Conclusion: Symptomatic patients with ipsilateral carotid stenosis and silent brain ischaemia on pre-operative MR-DWI, more often showed pathological evidence of IPH compared with those without ischaemic lesions. This identifies carotid IPH as a marker for patients at risk of silent brain ischaemia and possibly for future stroke and other arterial disease complications. Such patients may be more likely to benefit from CEA than those without evidence of ipsilateral carotid IPH

Primary tumor location (right left side of the colon) and resection affect the survival of patients with liver metastases from colonic neuroendocrine carcinoma: a population-based study

Background: Colonic neuroendocrine carcinomas (co-NECs) are heterogeneous and aggressive, especially with regard to metastasis. Whether co-NECs on the right and left sides of the colon have different characteristics from colon adenocarcinoma is unknown. Methods: The co-NEC patients were selected from the 2010–2017 Surveillance, Epidemiology, and End Results Program (SEER) database. The right and left sides of the colon were separated by the splenic flexure. Coarsened exact matching (CEM) was performed to adjust for relevant factors before regression models were constructed. Results: A total of 669 pathologically diagnosed co-NEC patients with sufficient baseline data were identified from the SEER database. A total of 80.72% of the patients had co-NEC that originated from the right side of the colon, and their mean overall survival (mOS) was similar to that of the patients with left-sided co-NECs (right versus left: 22.30 m versus 22.55 m). A total of 44.84% of the patients were diagnosed with liver metastasis (46.68% right side versus 37.98% left side). In patients with liver metastasis, those with right-sided co-NECs had better survival than those with left-sided co-NECs (mOS right versus left: 15.37 m versus 9.62 m; adjusted hazard ratio (HR) = 0.69, 95% confidence interval (CI): 0.49–0.98, p  = 0.035). To further investigate the survival benefits of primary site resection, we separated the patients who had liver metastasis according to the primary site and performed CEM to balance the groups (no patients underwent liver metastasis resection or intervention). The results suggested that primary surgery could benefit patients with both left- and right-sided co-NECs (adjusted HR = 0.50, 95% CI: 0.33–0.77, p  = 0.001 on the right side; HR = 0.38, 95% CI: 0.16–0.89, p  = 0.026 on the left side). Conclusions: Co-NECs frequently originate on the right side and commonly develop liver metastasis. Right-sided co-NECs are associated with better survival than left-sided co-NECs after liver metastasis has occurred. Primary site resection is associated with prolonged survival in co-NEC patients with liver metastasis, regardless of the side from which the co-NEC has originated