Tumour infiltrating lymphocyte status is superior to histological grade, DNA mismatch repair and BRAF mutation for prognosis of colorectal adenocarcinomas with mucinous differentiation.

Mucinous colorectal adenocarcinoma (CRC) is conventionally defined by extracellular mucin comprising >50% of the tumour area, while tumours with ≤50% mucin are designated as having a mucinous component. However, these definitions are largely arbitrary and comparisons of clinico-molecular features and outcomes by proportion of mucinous component are limited. A cohort of 1643 patients with stage II/III cancer was examined for tumour mucinous component, DNA mismatch repair (MMR) status, BRAF mutation and tumour infiltrating lymphocytes (TILs). Tumours with ≤50% mucinous component exhibited similar characteristics as mucinous tumours, including association with female gender, proximal location, high grade, TIL-high, defective MMR (dMMR) and BRAF mutation. Proportion of mucinous component did not stratify disease-free survival (DFS). In univariate analysis dMMR status, but not histological grade, stratified survival for mucinous and mucinous component tumours; however, in multivariate analysis dMMR status was not an independent predictor. BRAF mutation prognostic value depended on mucinous differentiation and MMR status, with poor prognosis limited to non-mucinous pMMR tumours (HR 2.61, 95% CI 1.69-4.03; p < 0.001). TIL status was a strong independent predictor of DFS in mucinous/mucinous component tumours (HR 0.40, 95% CI 0.23-0.67; p < 0.001), and a superior predictor of prognosis compared with histological grade, MMR and BRAF mutation. Mucinous component and mucinous stage II/III CRCs exhibit clinico-molecular resemblances, with histological grade and BRAF mutation lacking prognostic value. Prognosis for these tumours was instead strongly associated with TIL status, with the most favourable outcomes in TIL-high dMMR tumours, whilst TIL-low tumours had poor outcomes irrespective of MMR status.

Overexpression of TP53 protein is associated with the lack of adjuvant chemotherapy benefit in patients with stage III colorectal cancer.

TP53 mutations drive colorectal cancer development, with missense mutations frequently leading to accumulation of abnormal TP53 protein. TP53 alterations have been associated with poor prognosis and chemotherapy resistance, but data remain controversial. Here, we examined the predictive utility of TP53 overexpression in the context of current adjuvant treatment practice for patients with stage III colorectal cancer. A prospective cohort of 264 stage III patients was tested for association of TP53 expression with 5-year disease-free survival, grouped by adjuvant treatment. Findings were validated in an independent retrospective cohort of 274 stage III patients. Overexpression of TP53 protein (TP53+) was found in 53% and 52% of cases from the prospective and retrospective cohorts, respectively. Among patients receiving adjuvant chemotherapy, TP53+ status was associated with shorter disease-free survival (p ≤ 0.026 for both cohorts), while no difference in outcomes between TP53+ and TP53- cases was observed for patients treated with surgery alone. Considering patients with TP53- tumors, those receiving adjuvant treatment had better outcomes compared with those treated with surgery alone (p ≤ 0.018 for both cohorts), while no treatment benefit was apparent for patients with TP53+ tumors. Combined cohort-stratified analysis adjusted for clinicopathological variables and DNA mismatch repair status confirmed a significant interaction between TP53 expression and adjuvant treatment for disease-free survival (pinteraction = 0.030). For the combined cohort, the multivariate hazard ratio for TP53 overexpression among patients receiving adjuvant chemotherapy was 2.03 (95% confidence interval 1.41-2.95, p < 0.001), while the hazard ratio for adjuvant treatment among patients with TP53- tumors was 0.42 (95% confidence interval 0.24-0.71, p = 0.001). Findings were maintained irrespective of tumor location or when restricted to mismatch repair-proficient tumors. Our data suggest that adjuvant chemotherapy benefit in stage III colorectal cancer is restricted to cases with low-level TP53 protein expression. Identifying TP53+ tumors could highlight patients that may benefit from more aggressive treatment or follow-up.

Lymphocytic response to tumour and deficient DNA mismatch repair identify subtypes of stage II/III colorectal cancer associated with patient outcomes.

OBJECTIVE: Tumour-infiltrating lymphocyte (TIL) response and deficient DNA mismatch repair (dMMR) are determinants of prognosis in colorectal cancer. Although highly correlated, evidence suggests that these are independent predictors of outcome. However, the prognostic significance of combined TIL/MMR classification and how this compares to the major genomic and transcriptomic subtypes remain unclear. DESIGN: A prospective cohort of 1265 patients with stage II/III cancer was examined for TIL/MMR status and BRAF/KRAS mutations. Consensus molecular subtype (CMS) status was determined for 142 cases. Associations with 5-year disease-free survival (DFS) were evaluated and validated in an independent cohort of 602 patients. RESULTS: Tumours were categorised into four subtypes based on TIL and MMR status: TIL-low/proficient-MMR (pMMR) (61.3% of cases), TIL-high/pMMR (14.8%), TIL-low/dMMR (8.6%) and TIL-high/dMMR (15.2%). Compared with TIL-high/dMMR tumours with the most favourable prognosis, both TIL-low/dMMR (HR=3.53; 95% CI=1.88 to 6.64; Pmultivariate<0.001) and TIL-low/pMMR tumours (HR=2.67; 95% CI=1.47 to 4.84; Pmultivariate=0.001) showed poor DFS. Outcomes of patients with TIL-low/dMMR and TIL-low/pMMR tumours were similar. TIL-high/pMMR tumours showed intermediate survival rates. These findings were validated in an independent cohort. TIL/MMR status was a more significant predictor of prognosis than National Comprehensive Cancer Network high-risk features and was a superior predictor of prognosis compared with genomic (dMMR, pMMR/BRAFwt /KRASwt , pMMR/BRAFmut /KRASwt , pMMR/BRAFwt /KRASmut ) and transcriptomic (CMS 1-4) subtypes. CONCLUSION: TIL/MMR classification identified subtypes of stage II/III colorectal cancer associated with different outcomes. Although dMMR status is generally considered a marker of good prognosis, we found this to be dependent on the presence of TILs. Prognostication based on TIL/MMR subtypes was superior compared with histopathological, genomic and transcriptomic subtypes.

Validity and reliability of Ki-67 assessment in oestrogen receptor positive breast cancer.

Ki-67 is a prognostic and predictive biomarker in oestrogen receptor positive breast cancer. However, its measurement is not well standardised. This study compared the validity, intra- and inter-observer reproducibility and reporting time of five methods of Ki-67 assessment on tissue microarrays (TMA) and whole slides. Ki-67 labelling index (LI) was assessed on 71 breast carcinomas of no special type (NST), using five methods: manual counting (gold standard), unaided visual estimation, visual estimation aided by reference photographs, semi-manual digital image analysis (DIA) and fully automated DIA (Aperio platform). On TMA, semi-manual DIA demonstrated the closest agreement with the gold standard [intra-class correlation coefficient (ICC)=0.99 (95% confidence interval 0.98-0.99)]. All other methods also demonstrated close agreement [unaided estimation ICC=0.92 (0.90-0.93), aided estimation ICC=0.93 (0.92-0.95), fully automated DIA ICC=0.97 (0.96-0.97)]. On whole slides, both aided estimation and semi-manual DIA demonstrated excellent agreement with the gold standard [aided visual estimation ICC=0.91 (0.85-0.94), semi-manual DIA ICC=0.94 (0.89-0.96)]. Aided visual estimation significantly improved inter-observer reproducibility compared to unaided estimation [unaided ICC=0.87 (0.80-0.92); aided ICC=0.96 (0.93-0.97)] and corrected the underestimation bias seen in unaided estimation. Importantly, validity and reproducibility on whole slides were lower than on TMA for all methods of assessment, suggesting that field selection is an important source of variability in Ki-67 assessment. Values close to clinically used cut-off values therefore should be interpreted with caution.

Molecular profiling of cetuximab and bevacizumab treatment of colorectal tumours reveals perturbations in metabolic and hypoxic response pathways.

Angiogenesis and epidermal growth factor receptor (EGFR) inhibition has been shown to have anti-tumour efficacy, and enhance the therapeutic effects of cytotoxic chemotherapy in metastatic colorectal cancer. The interplay of signalling alterations and changes in metabolism and hypoxia in tumours following anti-VEGF and anti-EGFR treatment is not well understood. We aimed to explore the pharmacodynamics of cetuximab and bevacizumab treatment in human colon carcinoma tumour cells in vitro and xenograft models through proteomic profiling, molecular imaging of metabolism and hypoxia, and evaluation of therapy-induced changes in tumour cells and the tumour microenvironment. Both cetuximab and bevacizumab inhibited tumour growth in vivo, and this effect was associated with selectively perturbed glucose metabolism and reduced hypoxic volumes based on PET/MRI imaging. Global proteomic profiling of xenograft tumours (in presence of cetuximab, bevacizumab, and combination treatments) revealed alterations in proteins involved in glucose, lipid and fatty acid metabolism (e.g., GPD2, ATP5B, STAT3, FASN), as well as hypoxic regulators and vasculogenesis (e.g., ATP5B, THBS1, HSPG2). These findings correlated with western immunoblotting (xenograft lysates) and histological examination by immunohistochemistry. These results define important mechanistic insight into the dynamic changes in metabolic and hypoxic response pathways in colorectal tumours following treatment with cetuximab and bevacizumab, and highlight the ability of these therapies to selectively impact on tumour cells and extracellular microenvironment.

Tissue lipopolysaccharide-binding protein expression in rats after thermal injury: potential role of TNF-alpha.

Recent studies have suggested that levels of lipopolysaccharide-binding protein (LBP) might play a harmful role by up-regulating the host's sensitivity to endotoxin. Our previous studies demonstrated that local endotoxin could up-regulate LBP expression after acute insults, however, the definite molecular mechanisms downstream of endotoxin action remain unclear. This study investigates whether tumor necrosis factor (TNF-alpha) might be responsible for the LBP formation during endogenous endotoxemia postburn. Wistar rats were anesthetized, and a 35% TBSA full-thickness burn was created. Animals were randomly divided into normal control, thermal injury and anti-TNF-alpha mAb treatment group. A significant elevation of plasma endotoxin concentration was observed after acute insults. TNF-alpha levels in plasma also rapidly increased after thermal injury. Meanwhile, LBP mRNA expression markedly increased in liver, lungs, kidneys and intestine postburn. There was no detectable TNF-alpha in the plasma of anti-TNF-alpha mAb treated animals. Treatment with anti-TNF-alpha mAb also resulted in significantly lower concentrations of LBP mRNA in local tissues. Additionally, several organ function parameter levels in plasma significantly decreased in treatment group. These results demonstrated that an increase of plasma TNF-alpha levels caused by burns might be associated with a marked elevation of tissue LBP mRNA expression, which could contribute to the development of multiple organ damage.

Lipopolysaccharide-binding protein and lipopolysaccharide receptor CD14 gene expression after thermal injury and its potential mechanism(s).

BACKGROUND: We hypothesized that lipopolysaccharide-binding protein (LBP) and lipopolysaccharide receptor CD14 would present a pair of key molecules in pathophysiologic alterations induced by low concentrations of endotoxin after trauma. The aim of this study was to investigate the relationship between endotoxin translocation and tissue LBP/CD14 messenger ribonucleic acid (mRNA) expression after burn injury, and to define the potential role of LBP/CD14 in mediating inflammatory mediator induction, as well as the pathogenesis of organ damage. METHODS: Wistar rats were subjected to a 35% full-thickness scald injury, and tissue samples from liver, kidneys, lungs, and intestine were collected to measure LBP/CD14 and tumor necrosis factor-alpha (TNF-alpha) mRNA expression. Peritoneal macrophages were harvested by peritoneal lavage to determine CD14 mRNA expression. RESULTS: It was found that endotoxin levels in liver, spleen, and lung increased markedly after thermal injury, with the highest level in liver. Both tissue LBP and CD14 mRNA expression increased markedly after burns, peaking at 12 hours, and then decreasing gradually. At 48 hours, LBP gene expression had a tendency to the baseline level, whereas CD14 mRNA expression increased again. Likewise, CD14 mRNA levels were up-regulated markedly in peritoneal macrophages. Conversely, gene expression of TNF-alpha in tissues elevated markedly after acute insults. There were positive correlations between lipopolysaccharide levels and LBP/CD14 mRNA as well as TNF-alpha mRNA expression in tissues. Similar results were also obtained between CD14, TNF-alpha mRNA expression in liver tissue and liver function parameters, and between pulmonary TNF-alpha mRNA and myeloperoxidase activities (p < 0.01). CONCLUSION: Thermal injury per se can markedly up-regulate both LBP and CD14 gene expression in various organs. Excessive LBP and CD14 mRNA expression might be associated with enhanced synthesis and release of TNF-alpha stimulated by endotoxin translocation after major burns.