The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Demonstration of Translation Elongation Factor 3 Activity From a Non-Fungal Species, Phytophthora infestans

In most eukaryotic organisms, translation elongation requires two highly conserved elongation factors eEF1A and eEF2. Fungal systems are unique in requiring a third factor, the eukaryotic Elongation Factor 3 (eEF3). For decades, eEF3, a ribosome-dependent ATPase, was considered “fungal-specific”, however, recent bioinformatics analysis indicates it may be more widely distributed among other unicellular eukaryotes. In order to determine whether divergent eEF3-like proteins from other eukaryotic organisms can provide the essential functions of eEF3 in budding yeast, the eEF3-like proteins from Schizosaccharomyes pombe and an oomycete, Phytophthora infestans, were cloned and expressed in Saccharomyces cerevisiae. Plasmid shuffling experiments showed that both S. pombe and P. infestans eEF3 can support the growth of S. cerevisiae in the absence of endogenous budding yeast eEF3. Consistent with its ability to provide the essential functions of eEF3, P. infestans eEF3 possessed ribosome-dependent ATPase activity. Yeast cells expressing P. infestans eEF3 displayed reduced protein synthesis due to defects in translation elongation/termination. Identification of eEF3 in divergent species will advance understanding of its function and the ribosome specific determinants that lead to its requirement as well as contribute to the identification of functional domains of eEF3 for potential drug discovery

<i>P</i>. <i>infestans</i> eEF3 possesses ribosome-stimulated ATPase activity.

<p>ATPase activity of <i>S</i>. <i>cerevisiae</i> (■) and <i>P</i>. <i>infestans</i> (●) eEF3 was assayed in the presence (filled) or absence (empty) of <i>S</i>. <i>cerevisiae</i> ribosomes. The P_i released was measured using the PiColorLock Gold Phosphate Detection System. Reactions included varying amounts of eEF3, 25 nM ribosomes and 1 mM ATP and were carried out at room temperature for 30 min.</p

<i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i> and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 complement the loss of <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i> eEF3.

<p>(A) Yeast strains expressing eEF3 from the indicated species as the only form of eEF3 were streaked onto YEPD medium and incubated at 30°C for 2 d. CEN–low copy number plasmid/low expression. 2μ –high copy number plasmid/high expression. (B) Growth curves were generated from A₆₀₀ measurements of exponentially growing cultures over the indicated time period. Error bars represent standard error. (C) Whole cell extracts were prepared from the indicated strains, separated by SDS-PAGE, and stained with Ponceau S (bottom panel). The membrane was then cut and immunoblotted with either an anti-His antibody to detect eEF3 or anti-Pgk1 antibody as a loading control. The control lane is an extract from a yeast strain (TKY1617) that does not express epitope tagged eEF3.</p

<i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i> and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 complement the loss of <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i> eEF3.

<p>(A) Yeast strains expressing eEF3 from the indicated species as the only form of eEF3 were streaked onto YEPD medium and incubated at 30°C for 2 d. CEN–low copy number plasmid/low expression. 2μ –high copy number plasmid/high expression. (B) Growth curves were generated from A₆₀₀ measurements of exponentially growing cultures over the indicated time period. Error bars represent standard error. (C) Whole cell extracts were prepared from the indicated strains, separated by SDS-PAGE, and stained with Ponceau S (bottom panel). The membrane was then cut and immunoblotted with either an anti-His antibody to detect eEF3 or anti-Pgk1 antibody as a loading control. The control lane is an extract from a yeast strain (TKY1617) that does not express epitope tagged eEF3.</p

<i>S</i>. <i>cerevisiae</i> strains used in this study.

<p><i>S</i>. <i>cerevisiae</i> strains used in this study.</p

Strains expressing <i>P</i>. <i>infestans</i> eEF3 show defects in translation elongation and/or termination.

<p>(A) Antibiotic sensitivity was determined by measuring the diameter of the zone of growth inhibition around a disk containing 10 μL of the indicated drug: Paromomycin (800 mg/ml), hygromycin (25 mM), and cycloheximide (1 mM). The graph represents the average of three experiments and error bars representing the standard error are shown. (B) Ribosome extracts were prepared from the indicated strains in the absence of cycloheximide. Extracts were analyzed by 7–47% sucrose density gradient centrifugation and representative A₂₅₄ traces are shown. The area under the 80S and polyribosome peaks was analyzed using Image J and the ratio of polyribosomes to monosomes (p/m) is indicated for each strain. <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, <i>and P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i>.</p

Domain specific differences in the conservation of eEF3.

<p>(A) Individual domains of <i>S</i>.<i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 were aligned using Clustal Omega and the percentage identity to <i>S</i>. <i>cerevisiae</i> eEF3 is shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref023" target="_blank">23</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref024" target="_blank">24</a>]. The amino acids comprising each domain in <i>S</i>. <i>cerevisiae</i> are indicated. (B) Alignment of the chromodomain of <i>S</i>. <i>cerevisiae</i>, <i>S</i>. <i>pombe</i>, and <i>P</i>. <i>infestans</i> eEF3 was performed using Clustal Omega and shading based on identity was done using Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref025" target="_blank">25</a>]. Dark gray represents identity in all three species and light gray represents identity in two species. (C) Maximum likelihood tree with 500 bootstrap replicates created using phylogeny.fr.</p

<i>P</i>. <i>infestans</i> eEF3 possesses ribosome-stimulated ATPase activity.

<p>ATPase activity of <i>S</i>. <i>cerevisiae</i> (■) and <i>P</i>. <i>infestans</i> (●) eEF3 was assayed in the presence (filled) or absence (empty) of <i>S</i>. <i>cerevisiae</i> ribosomes. The P_i released was measured using the PiColorLock Gold Phosphate Detection System. Reactions included varying amounts of eEF3, 25 nM ribosomes and 1 mM ATP and were carried out at room temperature for 30 min.</p

Strains expressing <i>P</i>. <i>infestans</i> eEF3 exhibit a reduction in protein synthesis.

<p><i>S</i>. <i>cerevisiae</i> strains expressing eEF3 from the indicated species (<i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, <i>and P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> were grown to log phase in C-MET at 30°C. [³⁵S] methionine was added and total protein synthesis was measured by tricholoroacetic acid precipitation. Incorporation (counts per min) is expressed per A₆₀₀ unit. Each time point was performed in triplicate and error bars represent standard error. A representative experiment is shown.</p