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Background: The standard approach for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision (TME). After nCRT 20% of patients achieve a clinical complete response (pCR) and could be treated with a non-operative management (NOM). Methods: The panel of the Italian Association of Medical Oncology (AIOM) Guidelines on rectal cancer applied the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach addressing the following question: Should NOM vs. TME be used for patients with rectal cancer with clinical complete response after nCRT? Five outcomes were identified: disease-free survival (DFS), mortality, local recurrence, colostomy rate, and functional outcomes. Results: Nine studies were included in the analysis. A higher risk of disease recurrence was observed in the NOM group compared to the TME group (RR = 1.69, 95% CI 1.08, 2.64) on the other hand, we observed a slightly positive but not significant effect on mortality of NOM (RR = 0.82, 95% CI 0.46, 1.45). Patients in the NOM group were more likely to experience local recurrence (RR = 5.37, 95% CI 2.56, 11.27) and patients in the TME group were more likely to have a permanent colostomy (RR = 0.15, 95% CI 0.08, 0.29). Only one study evaluated functional outcomes. The overall certainty of evidence was rated as very low. Conclusions: NOM was found to correlate with a higher risk of local recurrence which did not translate in worse OS and a lower colostomy rate. Due to the paucity of evidences, no recommendations are possible. NOM remains an experimental treatment; thus, patients managed with NOM should be enrolled in clinical trials with a dedicated follow-up schedule.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins' sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25-35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

For hundreds of years, mankind has benefited from the natural metabolic processes of microorganisms to obtain basic products such as fermented foods and alcoholic beverages. More recently, microorganisms have been exploited for the production of antibiotics, vitamins and enzymes to be used in medicine and chemical industries. Additionally, several modern drugs, including those for cancer therapy, are natural products or their derivatives. Protists are a still underexplored source of natural products potentially of interest for biotechnological and biomedical applications. This paper focuses on some examples of bioactive molecules from protists and associated bacteria and their possible use in biotechnology.

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK – a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction: The incidence of anastomotic leak (AL) has not decreased over the past decades and some important grey areas remain in its definition, prevention, and management. The aim of this study was to reach a national consensus on the definition of AL and to identify key points to be applied in clinical practice. Methods: A 3-step modified Delphi method was used to establish consensus. Ten representative members of the major Italian surgical scientific societies with proven colorectal expertise were selected after a call to action. After a comprehensive literature search, each expert drew a list of evidence-based statements which were voted in round one by the scientific board. Panel members were asked to mark “totally disagree”, “partially agree” or “totally agree” for each statement and provide comments. The same voting method was used for round 2. Round 3 consisted of a final face-to-face meeting. Results: Thirty-three statements (clustered into 14 topics) were included in round 1. Following the third voting round, a final list of 16 items was formulated, which encompass the following 9 topics: AL definition, patient- and operative-related risk factors, prevention measures, bowel preparation, surgical technique, intraoperative assessment, early diagnosis, radiological diagnosis and management of specific patterns of AL. The overall response rate was 100% for all items in all the three rounds. Conclusions: This Delphi survey identified items that expert colorectal surgeons agreed were important to be applied in the prevention, diagnosis, and management of AL. This represents the first consensus involving all relevant national scientific societies, defining important and shared concepts in the diagnosis and management of AL.

Introduction: Transverse colon cancer (TCC) is poorly studied, and TCC cases are often excluded from large prospective randomized trials because of their complexity and their potentially high complication rate. The best surgical approach for TCC has yet to be established. The aim of this large retrospective multicenter Italian series is to investigate the advantages and disadvantages of both hemicolectomy and transverse colectomy in order to identify the best surgical approach. Materials and methods: This was a retrospective cohort study of patients with mid-transverse colon cancer treated with a segmental colon resection or an extended hemicolectomy (right or left) between 2006 and 2016 in 28 high-volume (more than 70 procedures/year) Italian referral centers for colorectal surgery. Results: The study included 1529 patients, 388 of whom underwent a segmental resection while 1141 underwent an extended resection. A higher number of complications has been reported in the segmental group than in the extended group (30.1% versus 23.6%; p 0.010). In 42 cases the main complication was the anastomotic leak (4.4% versus 2.2%; p 0.020). Recovery outcomes also showed statistical differences: time to first flatus (p 0.014), time to first mobilization (p 0.040), and overall hospital stay (p < 0.001) were significantly shorter in the extended group. Even if overall survival were similar between the groups (95.1% versus 97%; p 0.384), 3-year disease-free survival worsened after segmental resection (78.1% versus 86.2%; p 0.001). Conclusions: According to our results, an extended right colon resection for TCC seems to be surgically safer and more oncologically valid.

Biological systems show impressive adaptations at extreme environments. In extreme environments, directional selection pressure mechanisms acting upon mutational events often produce functional and structural innovations. Examples are the antifreeze proteins in Antarctic fish and their lack of hemoglobin, and the thermostable properties of TAQ polymerase from thermophilic organisms. During the past decade, more than 4000 organisms have been part of genome-sequencing projects. This has enabled the retrieval of information about evolutionary relationships among all living organisms, and has increased the understanding of complex phenomena, such as evolution, adaptation, and ecology. Bioinformatics tools have allowed us to perform genome annotation, crosscomparison, and to understand the metabolic potential of living organisms. In the last few years, research in bioinformatics has started to migrate from the analysis of genomic sequences and structural biology problems to the analysis of genotype-phenotype mapping. We believe that the analysis of multi-omic information, particularly metabolic and transcriptomic data of organisms living in extreme environments, could provide important and general insights into the how natural selection in an ecosystem shapes the molecular constituents. Here we present a review of methods with the aim to bridge the gap between theoretical models, bioinformatics analysis and experimental settings. The amount of data suggests that bioinformatics could be used to investigate whether the adaptation is generated by interesting molecular inventions.We therefore review and discuss the methodology and tools to approach this challenge.

Oxidative stress is a particularly severe threat to Antarctic marine polar organisms because they are exposed to high dissolved oxygen and to intense UV radiation. This paper reports the features of three superoxide dismutases from the Antarctic psychrophilic ciliate Euplotes focardii that faces two environmental challenges, oxidative stress and low temperature. Two out of these are Cu,Zn superoxide dismutases (named Ef-SOD1a and Ef-SOD1b) and one belongs to the Mn-containing group (Ef-SOD2). Ef-SOD1s and Ef-SOD2 differ in their evolutionary history, expression and overall structural features. Ef-SOD1 genes are expressed at different levels, with Ef-SOD1b mRNA 20-fold higher at the ciliate optimal temperature of growth (4 °C). All Ef-SOD enzymes are active at 4 °C, consistent with the definition of cold-adapted enzymes. At the same time, they display temperatures of melting in the range 50–70 °C and retain residual activity after incubation at 65–75 °C. Supported by data of molecular dynamics simulation, we conclude that the E. focardii SODs combine cold activity, local molecular flexibility and thermo tolerance.

Introduction: We retrospectively investigated the impact of number or complete absence of nodes retrieved on survival of patients with rectal cancer (RC) treated with neoadjuvant radiation-therapy (NAT). Methods: All patients with RC treated with NAT followed by curative surgery from 2000 to 2014 in 14 Italian referral Centres for Colorectal Surgery were enrolled. Information about number of nodes harvested, node ratio, type of radiation therapy schedule and tumour stage were recorded. Impact of number or complete absence of nodes retrieved on overall survival (OS) and on cumulative incidence of death for disease (CIDD) was assessed and factors influencing node yield were investigated. Results: In total, 1407 patients were included. Mean number of nodes retrieved was 12.9, while no lymph nodes were found in only 32 patients (2%, ypNnull). Definite nodal stage was ypN0 in 1001 patients (71%) and ypN+ in 372 patients (27%). In multivariable analysis ypNnull patients showed worse OS and CIDD compared to both ypN0 and ypN+. In ypN0 patients, number of nodes assessed, stratified in 4 groups (<5, 5–10, 11–15 and > 15), did not significantly influence OS and CIDD. Long-course radiation schedule and early T stages negatively affected node assessment. Conclusion: Complete absence of nodes assessed was associated with worse prognosis compared to node-negative and node-positive patients. In node-negative patients number of nodes was not associated to OS and CIDD. Based on data from this large population of irradiated RC, number of nodes assessed has no prognostic impact in node-negative patients.

Preoperative chemoradiotherapy is widely used to improve local control of disease, sphincter preservation and to improve survival in patients with locally advanced rectal cancer. Patients enrolled in the present study underwent preoperative chemoradiotherapy, followed by surgical excision. Response to chemoradiotherapy was evaluated according to Mandard’s Tumor Regression Grade (TRG). TRG 3, 4 and 5 were considered as partial or no response while TRG 1 and 2 as complete response. From pretherapeutic biopsies of 84 locally advanced rectal carcinomas available for the analysis, only 42 of them showed 70% cancer cellularity at least. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy D 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group and HFM1 and ASXL2 showed a positive trend. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. The present study confirmed the possibility that gene expression profiling combined with integrative computational biology is useful to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cancer.

Recent literature suggests that the benefit of adjuvant chemotherapy (aCT) for rectal cancer patients might depend on the response to neoadjuvant chemoradiation (CRT). Aim was to evaluate whether the effect of aCT in rectal cancer is modified by response to CRT and to identify which patients benefit from aCT after CRT, by means of a pooled analysis of individual patient data from 13 datasets. Patients were categorized into three groups: pCR (ypT0N0), ypT1-2 tumour and ypT3-4 tumour. Hazard ratios (HR) for the effect of aCT were derived from multivariable Cox regression analyses. Primary outcome measure was recurrence-free survival (RFS). One thousand seven hundred and twenty three (1723) (52%) of 3,313 included patients received aCT. Eight hundred and ninety eight (898) patients had a pCR, 966 had a ypT1-2 tumour and 1,302 had a ypT3-4 tumour. For 122 patients response, category was missing and 25 patients had ypT0N+. Median follow-up for all patients was 51 (0-219) months. HR for RFS with 95% CI for patients treated with aCT were 1.25(0.68-2.29), 0.58(0.37-0.89) and 0.83(0.66-1.10) for patients with pCR, ypT1-2 and ypT3-4 tumours, respectively. The effect of aCT in rectal cancer patients treated with CRT differs between subgroups. Patients with a pCR after CRT may not benefit from aCT, whereas patients with residual tumour had superior outcomes when aCT was administered. The test for interaction did not reach statistical significance, but the results support further investigation of a more individualized approach to administer aCT after CRT and surgery based on pathologic staging.