Commonalities and differences in the mutational signature and somatic driver mutation landscape across solid and hollow viscus organs.

Advances in sequencing have revealed a highly variegated landscape of mutational signatures and somatic driver mutations in a range of normal tissues. Normal tissues accumulate mutations at varying rates ranging from 11 per cell per year in the liver, to 1879 per cell per year in the bladder. In addition, some normal tissues are also comprised of a large proportion of cells which possess driver mutations while appearing phenotypically normal, as in the oesophagus where a majority of cells harbour driver mutations. Individual tissue proliferation and mutation rate, unique mutagenic stimuli, and local tissue architecture contribute to this highly variegated landscape which confounds the functional characterization of driver mutations found in normal tissue. In particular, our understanding of the relationship between normal tissue somatic mutations and tumour initiation or future cancer risk remains poor. Here, we describe the mutational signatures and somatic driver mutations in solid and hollow viscus organs, highlighting unique characteristics in a tissue-specific manner, while simultaneously seeking to describe commonalities which can bring forward a basic unified theory on the role of these driver mutations in tumour initiation. We discuss novel findings which can be used to inform future research in this field.

Biallelic FBXW7 knockout induces AKAP8-mediated DNA damage in neighbouring wildtype cells.

Colorectal cancer possesses marked intratumoral heterogeneity. While subclonal interactions between Vogelstein driver mutations have been extensively studied, less is known about competitive or cooperative effects between subclonal populations with other cancer driver mutations. FBXW7 is a cancer driver mutation which is present in close to 17% of colorectal cancer cells. In this study, we generated isogenic FBXW7 mutant cells using CRISPR-Cas9. We identified an upregulation of oxidative phosphorylation and DNA damage in FBXW7 mutant cells, which surprisingly proliferated at a decreased rate compared to wildtype cells. To determine subclonal interactions, wildtype and mutant FBXW7 cells were cocultured using a Transwell system. Wildtype cells cocultured with FBXW7 mutant cells similarly developed DNA damage which was not observed when wildtype cells were co-cultured with other wildtype cells, suggesting that FBXW7 mutant cells were inducing DNA damage in neighbouring wildtype cells. Using mass spectrometry, we identified AKAP8 as being secreted by FBXW7 mutant cells into the coculture media. Furthermore, overexpression of AKAP8 in wildtype cells recapitulated the DNA damage phenotype observed during coculture, while co-culture of wildtype cells with double mutant FBXW7-/-/AKAP8-/- cells abrogated the DNA damage phenotype. Here, we describe a hitherto unknown phenomenon of AKAP8-mediated DNA damage from FBXW7 mutant to neighbouring wildtype cells. Our findings demonstrate the importance of elucidating the local effect of cancer driver mutations between subclonal populations.

Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities.

Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.

Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9.

While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here, we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein.

Primary tumour immune response and lymph node yields in colon cancer.

BACKGROUND: The mechanism underlying improved survival in non-metastatic colon cancer with higher lymph node (LN) yield is unknown. This study aimed to identify whether molecular features in the primary tumour were predictive of LN yield. METHODS: Clinical, genomic, transcriptomic, proteomic and methylation data of non-metastatic, colon cancers studied in The Cancer Genome Atlas were interrogated for associations with LN yield. Based on maximal survival effects, patients were segregated into high (>15) and low (≤15) LN yield. Gene set enrichment analysis was performed on transcriptomic changes to identify biological processes associated with LN yield. Correlations were validated in an independent set of Stage II colon cancers. RESULTS: High LN yield was found predictive of overall and disease-free survival. There was no association of higher LN yield and increasing nodal positivity. High LN yield was strongly linked with gene expression changes associated with the adaptive and dendritic cell immune response. This association was most prominent in node-negative cancers. Analogous findings were reproduced in the validation dataset. CONCLUSION: The study shows a strong association of an activated immune response in tumours with a high LN yield. Immunogenic tumours have a better prognosis, likely explaining the survival benefit with higher LN yields.

Tumour heterogeneity and evolutionary dynamics in colorectal cancer.

Colorectal cancer (CRC) has a global burden of disease. Our current understanding of CRC has progressed from initial discoveries which focused on the stepwise accumulation of key driver mutations, as encapsulated in the Vogelstein model, to one in which marked heterogeneity leads to a complex interplay between clonal populations. Current evidence suggests that an initial explosion, or "Big Bang", of genetic diversity is followed by a period of neutral dynamics. A thorough understanding of this interplay between clonal populations during neutral evolution gives insights into the roles in which driver genes may participate in the progress from normal colonic epithelium to adenoma and carcinoma. Recent advances have focused not only on genetics, transcriptomics, and proteomics but have also investigated the ecological and evolutionary processes which transform normal cells into cancer. This review first describes the role which driver mutations play in the Vogelstein model and subsequently demonstrates the evidence which supports a more complex model. This article also aims to underscore the significance of tumour heterogeneity and diverse clonal populations in cancer progression.

Perioperative and oncologic outcomes in young and octogenarian patients with colorectal cancer: a comparison at the extremes.

INTRODUCTION: In colorectal cancer (CRC), surgical outcomes in the young (< 50) and octogenarian populations are believed to be poor due to an aggressive phenotype in the former, and increased frailty in the latter. Given that age-related effects are inversely related between groups, we compared short- and long-term outcomes of young and octogenarian patients with CRC to determine the dominance of one age-related factor over another. METHODS: A prospectively collected database from 2015 to 2020 of all CRC was analyzed. Cases were divided into metastatic and non-metastatic groups. RESULTS: Among non-metastatic cases, there were 34 young and 113 octogenarian patients. Mean CEA was higher among octogenarians (11.2 vs 32.8 units/ml; p = 0.041). Octogenarians suffered from more comorbidities than younger counterparts, with increased rates of postoperative UTI (3.3% vs 10.3%; p = 0.246) and pneumonia (3.1% vs 8.8%; p = 0.331). There was no increased rate of reintervention or Clavien-Dindo scores. We noticed a statistically significant higher proportion of extramural vascular invasion (EMVI) (8.8% vs 32.3%; p = 0.003) among the young. When excluding octogenarians who had declined surgery, Kaplan-Meier analysis showed no difference in disease-free (p = 0.290) or overall survival (p = 0.111). Among metastatic cases, there were 21 young and 19 octogenarian patients. Young patients were treated more aggressively with chemotherapy (55.6% vs 12.5%; p = 0.040). There was however no difference in overall survival between groups (p = 0.610). CONCLUSIONS: Octogenarians may have more comorbidities, but can achieve similar surgical outcomes with younger patients. There is no reason to suspect a more aggressive phenotype in younger patients.

Chemotherapy and adverse cardiovascular events in colorectal cancer patients undergoing surgical resection.

BACKGROUND: Colorectal cancer patients undergoing surgical resection are at increased short-term risk of post-operative adverse events. However, specific predictors for long-term major adverse cardiac and cerebrovascular events (MACCE) are unclear. We hypothesised that patients who receive chemotherapy are at higher risk of MACCE than those who did not. METHODS: In this retrospective study, 412 patients who underwent surgical resection for newly diagnosed colorectal cancer from January 2013 to April 2015 were grouped according to chemotherapy status. MACCE was defined as a composite of cardiovascular death, myocardial infarction, stroke, unplanned revascularisation, hospitalisation for heart failure or angina. Predictors of MACCE were identified using competing risks regression, with non-cardiovascular death a competing risk. RESULTS: There were 200 patients in the chemotherapy group and 212 patients in the non-chemotherapy group. The overall prevalence of prior cardiovascular disease was 20.9%. Over a median follow-up duration of 5.1 years from diagnosis, the incidence of MACCE was 13.3%. Diabetes mellitus and prior cardiovascular disease were associated with an increased risk of MACCE (subdistribution hazard ratio, 2.56; 95% CI, 1.48-4.42) and 2.38 (95% CI, 1.36-4.18) respectively. The chemotherapy group was associated with a lower risk of MACCE (subdistribution hazard ratio, 0.37; 95% CI, 0.19-0.75) compared to the non-chemotherapy group. CONCLUSIONS: Amongst colorectal cancer patients undergoing surgical resection, there was a high incidence of MACCE. Diabetes mellitus and prior cardiovascular disease were associated with an increased risk of MACCE. Chemotherapy was associated with a lower risk of MACCE, but further research is required to clarify this association.

Continuous wound infusion with ropivacaine alone provides adequate analgesia following laparotomy.

BACKGROUND: Although continuous wound infusion (CWI) with local anaesthetic has been used as an adjunct for pain relief following laparotomy, its use as the main modality has not been studied. This approach negates side effects related to intravenous opioid administration, therefore promoting enhanced recovery from surgery. We conducted this study to investigate the feasibility and efficacy of CWI following laparotomy. METHODS: Consecutive patients who underwent laparotomy from June 2016 to December 2019 were analysed. All patients were given CWI with only oral supplementation. Pain was assessed based on the numeric rating scale (NRS). RESULTS: One hundred and three patients were analysed. Mean age was 61.1 (standard deviation 16.7). 47.6% of patients were operated for intestinal obstruction. Large bowel resection was the most common operation performed (49.5%). 69.9% of patients underwent emergency surgery, whilst 51.5% of patients had surgery for cancer. On postoperative day 0, NRS was 3.2 (standard deviation (sd) 2.6) which decreased to 1.5 (sd 1.9) on day 3, and 1.1 (sd 1.8) on day 5. Mean time to flatus was 2.3 (sd 1.4) days, whilst mean time to first bowel movement was 3.1 (sd 1.7) days. Patients were able to commence ambulation by 2.5 (sd 1.8) days. Patients could tolerate a normal diet on day 3.9 (sd 3.3), and IV drip was removed on day 3.5 (sd 3.0). Mean length of stay was 9.1 (sd 6.9) days. Only two patients suffered from respiratory depression (1.9%) whilst five patients suffered from hypotension (4.9%). No patients had pruritus. 23.3% of patients had nausea or vomiting. Only one patient had a catheter-related complication which was easily addressed. CONCLUSION: CWI provides adequate pain relief as the principle modality of analgesia after surgery, without opioid side effects.

Early surgery after bridge-to-surgery stenting for malignant bowel obstruction is associated with better oncological outcomes.

BACKGROUND: Placement of self-expanding metal stents has been increasingly adopted as a bridge to surgery in patients presenting with obstructed left-sided colorectal cancers. The optimal bridging time has yet to be widely established, hence this retrospective study aims to determine the optimal bridging time to elective surgery post endoluminal stenting. PATIENTS AND METHODS: All patients who underwent colorectal stenting for large bowel obstruction in a single, tertiary hospital in Singapore between January 2003 and December 2017 were retrospectively identified. Patients' baseline demographics, tumour characteristics, stent-related complications, intra-operative details, post-operative complications and oncological outcomes were analysed. RESULTS: Of the 53 patients who successfully underwent colonic stenting for malignant left sided obstruction, 33.96% of patients underwent surgery within two weeks of stent placement while 66.04% of patients underwent surgery after 2 weeks of stent placement. Univariate analysis between both groups did not demonstrate significant differences in postoperative complications and stoma formation. Significant differences were observed between both groups for stent complications (38.89% vs 8.57%, p = 0.022), on-table decompression (38.89% vs 2.86%, p = 0.001) and systemic recurrence (11.11% vs 40.00%, p = 0.030). Increased bridging interval to surgery (OR 13.16, CI 1.37-126.96, p = 0.026) was a significant risk factor for systemic recurrence on multivariate analysis. CONCLUSIONS: Patients undergoing definitive surgery within 2 weeks of colonic stenting may have better oncological outcomes without compromising on postoperative outcomes. Further prospective studies are required to compare outcomes between emergency surgery and different bridging intervals.

Enhanced recovery program versus conventional care after colorectal surgery in the geriatric population: a systematic review and meta-analysis.

INTRODUCTION: Enhanced recovery after surgery (ERAS) programs for patients undergoing colorectal surgery has yielded promising results. However, there remains controversy regarding the application of ERAS protocols in an elderly population. The aim of this review is to compare the clinical outcomes between ERAS versus conventional peri-operative care (Non-ERAS) for colorectal surgery in patients aged ≥ 65 years old. METHODS: The PRISMA guidelines were adhered to. A comprehensive search was performed using Medline, Embase and the Cochrane Library electronic databases and relevant articles were identified. Indications for the colorectal resections included both benign and malignant diseases, while emergency surgeries were excluded. Primary outcomes include post-operative morbidity, length of stay and re-admission rates. Data analysis was performed using Revman 5.3. RESULTS: A total of six studies were included, which involved a total of 1174 patients. ERAS was associated with a lower incidence of post-operative morbidity compared to Non-ERAS (OR 0.38, 95% CI 0.25-0.59), p < 0.001). Similarly, ERAS was also associated with a significantly shorter hospital length of stay (MD - 2.49, 95% CI - 4.11 to 0.88, p = 0.002). Return of bowel function as measured by time to flatus was significantly faster in the ERAS group (MD - 20.01 95% CI - 36.23 to 3.79, p = 0.02), but post-operative ileus rates (OR 0.86, 95% CI 0.50-1.47, p = 0.58) were comparable. Re-admission, re-operation and post-operative mortality rates were also similar between both groups. CONCLUSION: The application of ERAS protocols in an elderly population provides the advantages of lower post-operative morbidity and shorter hospital length of stay. Future studies should aim to evaluate factors that can improve ERAS compliance rates in this group of patients.

Lymphocyte-White Cell Ratio Is a Novel Marker of Morbidity Following Colorectal Cancer Surgery.

BACKGROUND: A preoperative marker for morbidity in patients with colorectal cancer would help to risk stratify patients and allow for timely intervention to avert poor outcomes. We conducted this study to evaluate preoperative lymphocyte-white blood cell ratio (LWR) as a marker of postoperative morbidity. METHODS: A prospective cohort of patients who underwent elective surgery for colorectal cancer was reviewed. Three morbidity-related outcomes were described-overall morbidity, multiple morbidities, and severe morbidity, defined as Clavien-Dindo Class ≥3. Univariable and multivariable analyses of presurgical predictors of these three outcomes were performed. Preoperative variables included hemoglobin levels, neoadjuvant therapy, albumin levels, white blood cell count, lymphocyte count, LWR, neutrophil-lymphocyte ratio, and prognostic nutritional index. RESULTS: Of 177 patients, 31.6% (56/177) suffered at least one morbidity, 15.3% (27/177) had multiple morbidities, 7.9% (14/177) suffered severe morbidity. On multivariate analysis, only LWR <0.180 (odds ratio [OR] 2.53, 95% confidence interval [CI] 1.15-5.55) and neoadjuvant therapy (OR 2.49, 95% CI 1.16-5.24) were associated with overall morbidity. For multiple morbidities and severe morbidity, only LWR <0.180 was significantly associated on multivariate analysis with an OR of 2.92 (95% CI 1.19-7.13) and 4.62 (95% CI 1.45-14.73), respectively. CONCLUSIONS: LWR is a preoperative marker which can be conveniently applied using standard preoperative blood tests. LWR is an independent risk factor for overall morbidity, multiple morbidities, as well as severe morbidity when used with a cut-off of LWR<1.80.

A simple prediction score for prolonged length of stay following elective colorectal cancer surgery.

BACKGROUND: Current enhanced recovery after surgery (ERAS) protocols are designed for all patients without tailored programmes for at-risk groups. A risk score to determine elective colorectal cancer patients at risk for prolonged length of stay (LOS) would help to identify this group for preoperative intervention. METHODS: Multivariate analysis of demographic and preoperative variables was performed to identify independent risk factors for prolonged LOS, defined as 7 days or more. A stepwise variable selection approach using logistic regression was then used to build a risk prediction model. RESULTS: Among 172 patients in our population, 41.9% of patients had prolonged LOS. Five variables were included in our risk prediction model. These were age ≥ 65 years (OR 13.9 5.09-38.0; p < 0.0001), neoadjuvant therapy (OR 7.60 2.51-23.0; p < 0.0001), open approach (OR 3.96 1.68-15.9); p = 0.008), history of smoking (OR 5.18 1.68-15.9; p = 0.004) and white blood cell (WBC) count (OR 0.83/unit 0.69-0.99; p = 0.040). These variables were combined to produce a score, for which the area under the receiving operator curve was 0.82 (95% CI 0.76-0.88), and Hosmer-Lemeshow test showed a χ2 statistic of 9.14 and p = 0.519. Using 0.9 as a cut-off, the score has sensitivity of 81.9% and specificity of 65.0%. CONCLUSION: A simple, clinical score can be used to predict for prolonged LOS based on preoperative variables, allowing for intervention before surgery. Age, neoadjuvant therapy, smoking status, open approach and WBC count are independent risk factors for prolonged length of stay following elective colorectal cancer surgery. A risk score comprising the above independent variables was developed with area under the receiving operator curve of 0.82 (95% CI 0.76-0.88), and a Hosmer-Lemeshow test showing a χ2 statistic of 9.14 and p = 0.519.

Indocyanine green fluorescence angiography decreases the risk of colorectal anastomotic leakage: Systematic review and meta-analysis.

BACKGROUND: Anastomotic leakage after a colorectal resection results in devastating consequences for patients. Indocyanine green fluorescence angiography is a modality to visualize vascular perfusion at the anastomotic site and can help surgeons decide the viability of the anastomosis. We performed this systematic review and meta-analysis to evaluate the efficacy of indocyanine green fluorescence angiography in decreasing anastomotic leakage. METHODS: PubMed, Web of Science, Embase, and the Cochrane Library were searched to identify studies comparing the use of indocyanine green fluorescence angiography versus standard care on rates of anastomotic leakage. Data were pooled with the Mantel-Haenszel method and analyzed based on a random-effects model to estimate the pooled odds ratio and 95% confidence interval. The heterogeneity of studies was evaluated using I2 statistic. RESULTS: Twenty studies were included in this meta-analysis of 5,498 patients. The pooled estimate of the odds ratio was 0.46 (95% confidence interval 0.34-0.62; P < .00001) favoring indocyanine green fluorescence angiography. The overall anastomotic leak rate was 3.7% (n = 82) in the intervention group and 8.6% (n = 282) in the control group. Indocyanine green fluorescence angiography led to a change in the anastomotic site in 216 (9.7%) patients. Subgroup analyses of anastomotic leakage requiring intervention, patients requiring a low colorectal anastomosis, and prospective studies had a pooled estimate of odds ratio 0.55 (95% confidence interval 0.35-0.89), odds ratio 0.38 (95% confidence interval 0.27-0.54; P < .0001), and odds ratio 0.49 (95% confidence interval 0.30-0.81; P = .005) respectively. CONCLUSION: The use of indocyanine green fluorescence angiography is associated with a decrease in anastomotic leakage. This association is present in patients with severe anastomotic leakage requiring intervention as well as low colorectal anastomoses.

Stenting versus surgery in obstructed malignant colorectal cancer-a review of short and long-term results.

Obstructed colorectal cancers (CRCs) continue to be challenging to manage. Apart from surgical resection, the use of self-expanding metallic stents (SEMS) has become increasingly popular. SEMS could potentially allow for the conversion of an emergency operation into a semi-elective procedure, allow for the optimization of the patient's physiology, avoid the requirement for a stoma, as well as allow for a minimally-invasive approach to be adopted. This review article therefore highlights the evidence available in the literature pertaining to the use of SEMS in obstructed CRCs.

Age is an independent risk factor for increased morbidity in elective colorectal cancer surgery despite an ERAS protocol.

INTRODUCTION: Elderly patients with colorectal cancer are high-risk surgical candidates. ERAS protocols have been developed to mitigate against these risks. We performed this study to quantify the risks which elderly patients face and then to determine independent risk factors for short-term ERAS-specific outcomes. METHODS: An analysis of a prospectively collected audit database of all patients who underwent elective colorectal cancer resection within an ERAS framework from January 2018 to December 2018 was performed. Elderly was defined in our study as age ≥ 65 years. RESULTS: There were 172 elective colorectal cancer resections performed. Ninety-seven (56.4%) were elderly. Elderly patients were at increased risk of developing post-operative complications (33.0% vs 16.0%, p = 0.011), longer time to diet (3.4 vs 2.2 days, p = 0.001), and longer hospital stay (10.9 vs 6.7 days, p = 0.007). Independent risk factors were determined for the abovementioned three outcomes. Elderly status was the only risk factor for increased complications (OR 2.61 95% CI (1.05-6.51), p = 0.040). For delayed time to soft diet, male gender (OR 6.67(1.92-20.0), p = 0.002), open approach (OR 9.06(2.26-36.30), p = 0.002), and increased operative time (OR 1.01(1.00-1.01) p = 0.014) were risk factors. Finally, elderly age (OR 5.53(1.82-16.84), p = 0.003), leucocyte count (OR 1.39(0.76-2.57), p = 0.038), open approach (OR 5.26(1.41-19.62), p = 0.013), operative time (OR 1.01(1.00-1.01), p = 0.021), and Clavien-Dindo classification (OR 7.97(1.27-49.88), p = 0.027) were risk factors for prolonged length of stay. CONCLUSION: Elderly patients are intrinsically at risk for increased complications, longer time to soft diet and longer hospital stay. ERAS protocols may need to be specifically tailored for elderly patients.