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Excel spreadsheet of NUAK1 inhibitor induced phospho-peptide alterations

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Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B−/− mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia.Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75–87. ©2017 AACR.

Supplemental Figure 1 (SF1) describes lifespan in global PTP1B-/- mice. SF2 shows development of leukemia is associated with myeloid PTP1B complete deficiency. SF3 shows tumors in LysM PTP1B-/- mice. SF4 presents LysM PTP1B-/- microarray data. SF5 presents STAT5 expression data. SF6 presents additional STAT3 ChIP seq data from LysM PTP1B mice. Tables 1-4 demonstrate detailed statistics for survival data on LysM PTP1B and global PTP1B-/- mice as well as hepatic pre-tumour microarray data from LysM PTP1B-/- and control mice.

PDF file - 100K, Primer sequences and detailed descriptions of immunohistochemistry, HUVEC wound closure, immunoblotting and in-vivo sponge assays. Includes supplemental references.

PDF file - 54K, Distribution of CD31 and LOX immunoreactivity in a tissue microarray of patient samples

PDF file - 574K, Endomucin staining in HT29 and LS174T subcutaneous tumors (S1); VEGF transcription and secretion in HT29 and LS174T cell lines and subcutaneous tumors (S2); Dependence of VEGF transcription and secretion on Akt(Ser473) phosphorylation (S3); Phosphorylation of PDGFRβ in response to LOX, and effect of PDGFRβ inhibition on VEGF transcription (S4); Effect of VEGF signaling inhibition on LOX-stimulated endothelial cell wound closure and sprouting (S5); In vivo sponge assay using SW620 conditioned media, and VEGF and CD31 immunostaining in a tissue microarray of patient samples (S6); Validation in the 4T1 breast cancer model (S7)

New treatment targets are needed for colorectal cancer (CRC). We define expression of High Mobility Group Box 1 (HMGB1) protein throughout colorectal neoplastic progression and examine the biological consequences of aberrant expression. HMGB1 is a ubiquitously expressed nuclear protein that shuttles to the cytoplasm under cellular stress. HMGB1 impacts cellular responses, acting as a cytokine when secreted. A total of 846 human tissue samples were retrieved; 6242 immunohistochemically stained sections were reviewed. Subcellular epithelial HMGB1 expression was assessed in a CRC Tissue Microarray ( n = 650), normal colonic epithelium ( n = 75), adenomatous polyps ( n = 52), and CRC polyps (CaP, n = 69). Stromal lymphocyte phenotype was assessed in the CRC microarray and a subgroup of CaP. Normal colonic epithelium has strong nuclear and absent cytoplasmic HMGB1. With progression to CRC, there is an emergence of strong cytoplasmic HMGB1 ( p < 0.001), pronounced at the leading cancer edge within CaP ( p < 0.001), and reduction in nuclear HMGB1 ( p < 0.001). In CRC, absent nuclear HMGB1 is associated with mismatch repair proteins ( p = 0.001). Stronger cytoplasmic HMGB1 is associated with lymph node positivity ( p < 0.001) and male sex ( p = 0.009). Stronger nuclear ( p = 0.011) and cytoplasmic ( p = 0.002) HMGB1 is associated with greater CD4 + T-cell density, stronger nuclear HMGB1 is associated with greater FOXP3 + ( p < 0.001) and ICOS + ( p = 0.018) lymphocyte density, and stronger nuclear HMGB1 is associated with reduced CD8 + T-cell density ( p = 0.022). HMGB1 does not directly impact survival but is associated with an 'immune cold' tumour microenvironment which is associated with poor survival ( p < 0.001). HMGB1 may represent a new treatment target for CRC.

Colorectal cancer (CRC) remains a leading cause of cancer mortality. Here, we define the colonic epithelial expression of cathelicidin (LL‐37) in CRC. Cathelicidin exerts pleotropic effects including anti‐microbial and immunoregulatory functions. Genetic knockout of cathelicidin led to increased size and number of colorectal tumours in the azoxymethane‐induced murine model of CRC. We aimed to translate this to human disease. The expression of LL‐37 in a large (n = 650) fully characterised cohort of treatment‐naïve primary human colorectal tumours and 50 matched normal mucosa samples with associated clinical and pathological data (patient age, gender, tumour site, tumour stage [UICC], presence or absence of extra‐mural vascular invasion, tumour differentiation, mismatch repair protein status, and survival to 18 years) was assessed by immunohistochemistry. The biological consequences of LL‐37 expression on the epithelial barrier and immune cell phenotype were assessed using targeted quantitative PCR gene expression of epithelial permeability (CLDN2, CLDN4, OCLN, CDH1, and TJP1) and cytokine (IL‐1β, IL‐18, IL‐33, IL‐10, IL‐22, and IL‐27) genes in a human colon organoid model, and CD3+, CD4+, and CD8+ lymphocyte phenotyping by immunohistochemistry, respectively. Our data reveal that loss of cathelicidin is associated with human CRC progression, with a switch in expression intensity an early feature of CRC. LL‐37 expression intensity is associated with CD8+ T cell infiltrate, influenced by tumour characteristics including mismatch repair protein status. There was no effect on epithelial barrier gene expression. These data offer novel insights into the contribution of LL‐37 to the pathogenesis of CRC and as a therapeutic molecule.

Patients with screened detected colorectal cancer (CRC) have a better survival than patients referred with symptoms. This may be because of cancers being identified in a younger population and at an earlier stage. In this study, we assess whether screened detected CRC has an improved outcome after controlling for key pathological and patient factors known to influence prognosis.This is a cohort study of all CRC patients diagnosed in NHS Grampian. Patients aged 51-75 years old between June 2007 and July 2017 were included. Data were obtained from a prospectively maintained regional pathology database and outcomes from ISD records. All-cause mortality rates at 1 and 5 years were examined. A Cox proportional hazards regression model was used to estimate the effect of screening status, age, gender, Duke stage, tumour location, extramural venous invasion (EMVI) status and lymph node ratio (LNR) on overall survival.Of 1618 CRC cases, 449 (27.8%) were screened and 1169 (72.2%) were symptomatic. Screened CRC patients had improved survival compared to non-screened CRC at 1 year (88.9% vs 83.9% p 0.001) and 5-years (42.5% vs 36.2%; p 0.001). On multivariable analysis of patients who had no neoadjuvant therapy (n = 1272), screening had better survival (HR 0.57; 95% CI 0.44-0.74; p 0.001). EMVI (HR 2.22; CI 1.76 to 2.79; p 0.001) and tumour location were found to affect outcome.Patients referred through screening had improved survival compared with symptomatic patients. Further research could be targeted to determine if screened CRC cases are pathologically different to symptomatic cancers or if the screening cohort is inherently more healthy.

Purpose Oesophageal squamous cell carcinoma (ESCC) has a poor prognosis. Advanced tumours are treated with fluoropyrimidine/platinum chemotherapy followed by irinotecan or taxane monotherapy, but resistance is common and new treatments are needed. Approximately 20% of ESCCs carry copy number gain (CNG) of the epidermal growth factor receptor (EGFR) gene. Previous trials show that while anti-EGFR monotherapy benefits biomarker-selected patients with EGFR CNG and/or high EGFR expression, combining anti-EGFR therapies with platinum fluoropyrimidine chemotherapies is not effective, and uncertainty remains regarding the optimal cytotoxic chemotherapy partner for anti-EGFR therapies in ESCC. Methods The effects of EGFR CNG on fluoropyrimidine/platinum chemotherapy sensitivity in a cohort of gastroesophageal cancer patients (n = 302) was evaluated. Drug combination studies using the EGFR inhibitor gefitinib with cytotoxic chemotherapies, docetaxel, cisplatin, oxaliplatin and irinotecan, on cell proliferation and cell death of EGFR CNG ESCC cell lines were assessed. Results EGFR CNG in gastroesophageal cancer patients was associated with improved overall survival following fluoropyrimidine/platinum chemotherapy. However, co-administration of gefitinib and oxaliplatin or cisplatin was frequently antagonistic in cell-based assays in EGFR CNG ESCC, whereas the combination of gefitinib with docetaxel or irinotecan was more efficacious. Co-administration of gefitinib/docetaxel and sequential administration of docetaxel before gefitinib showed synergy, but docetaxel given after gefitinib was antagonistic. Conclusion Gefitinib/platinum co-administration demonstrated antagonism suggesting a possible explanation for the lack of benefit from addition of anti-EGFR therapies to fluoropyrimidine/platinum chemotherapy in trials. Gefitinib/docetaxel co-administration demonstrated synergy suggesting taxanes could be the most effective cytotoxic partner for anti-EGFR therapies in EGFR CNG-positive ESCC, but careful consideration of drug scheduling is required.

Introduction: Colorectal cancer risk stratification traditionally focuses on tumor node metastasis staging. Seemingly equivalent tumors can differ unpredictably in prognosis; more sophisticated quantification and stratification methods are required to identify tumors with a high likelihood of becoming metastatic. Hypoxia (low oxygen concentration) is associated with aggressive phenotypes and poor prognosis. Hypoxia is associated with treatment resistance consequently there is an unmet clinical requirement to develop personalised treatment based on hypoxia. Positron emission tomography/computed tomography (PET/CT) imaging can non-invasively detect hypoxic tumors. [18F]Fluoroazomycin arabinoside ([18F]FAZA) is a leading hypoxia PET/CT radiotracer, and uptake is associated with lower disease free survival. Case Report: A 78-year-old man, diagnosed with a localised colorectal cancer, underwent [18F]FAZA PET/CT imaging pre-operatively. This confirmed hypoxic regions in the tumor with correlation demonstrated with carbonic anhydrase IX (CAIX) immunohistochemistry (IHC). He underwent a right hemicolectomy. The pathological staging for his colorectal cancer predicted a good outcome; thus, he did not receive adjuvant chemotherapy. The patient subsequently developed early metastatic disease with two lung metastases, which were resected by thoracotomy and wedge resection. He continues on follow up at present with no evidence of recurrent disease. Conclusion: Hypoxia can be an important marker in colorectal cancer when determining risk and prognosis. We present evidence of clinical correlation of FAZA uptake and CAIX IHC in colorectal cancer, a key aspect in FAZA tracer validation. PET/CT potentially provides a specific tool for stratification for hypoxia-related treatment modification and development of hypoxia biomarkers.

Background The incidence of oesophageal adenocarcinoma is increasing globally. Barrett’s oesophagus (BO) is a pre-malignant condition with no biomarker to risk stratify those at highest risk of dysplasia and malignant transformation. Methods Subcellular epithelial protein (HMGB1, p53, RUNX3) expression, alongside expression of CD20, CD4, CD8 and Foxp3 to characterise stromal B lymphocyte, and helper, cytotoxic and regulatory T-lymphocyte cell infiltrate, respectively, was assessed by immunohistochemistry in 218 human tissue samples including normal oesophageal/gastric biopsies (n = 39), BO (non-dysplasia, dysplasia, non-dysplastic background from progressors to dysplasia or cancer, n = 121) and oesophageal adenocarcinoma (n = 58). Results There is a dynamic subcellular epithelial expression of HMGB1 (loss of nuclear, emergence of cytoplasmic), associated with epithelial p53 expression and differential immune cell phenotype in oesophageal neoplastic progression. We identify a protein signature and lymphocyte infiltrate in non-dysplastic BO when progressive disease (dysplasia or adenocarcinoma) is present but not histologically represented in the biopsied field. There is a dynamic stromal lymphocytic infiltrate in oesophageal neoplastic progression. Conclusions This data reveals novel insights into the microenvironment of BO and progression towards cancer and identifies a novel high-risk biomarker of disease progression to aid surveillance strategies to identify early progression and impact future incidence of oesophageal cancer.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer related death. The aberrant expression of a brown fat-like phenotype in cancer cells has been previously implicated in tumour growth. Therefore, the expression of brown fat-associated proteins in colorectal cancer could be associated with tumour prognosis. Monoclonal antibodies to brown fat-associated proteins CIDEA, ELOVL3, ELOVL5, and UCP1 were developed. The antibodies were used to profile the expression of protein targets by immunohistochemistry in a discovery cohort comprising 50 normal colonic mucosa samples and 274 primary colorectal cancers and a validation cohort comprising 549 colorectal cancers. Immunostaining for UCP1 was observed in the majority of colorectal tumours while no immunostaining was observed in normal colonic mucosa (p

Aim: Neoadjuvant treatment (NaT) for locally advanced rectal cancer prior to surgery has led to improved outcomes. However, the relationship between pathological response to NaT and survival is not entirely clear. The aim of this study was to assess the degree of pathological response to NaT on survival outcomes. Methods: Clinical and pathological data were collected from a prospectively maintained pathology database between 2005 and 2017. The primary outcome was the overall survival based on pathological response categorized as complete, good partial, partial and minimal. Univariate and multivariate analyses were conducted to identify variables predictive of survival. Cox proportional hazard ratios were used for survival. Results: A total of 596 patients had surgery following NaT for locally advanced rectal cancer. The median follow-up was 4.57 years (interquartile range 2.21–8.15 years). The overall survival for complete pathological response was 75.6% vs. 37.3% for minimal response (P [less than] 0.001). The overall survival at the end of the study in the good partial vs. partial response groups was 58.9% vs. 39% (P [less than] 0.001). On multivariate analysis, the degree of pathological response remains an independent variable for overall and disease-specific survival across all categories. Discussion: In addition to other pathological variables, the degree of pathological response to NaT is an independent predictor for survival outcomes. Future verification of these findings elsewhere could support NaT response being used for adjuvant therapy decision making.

Objective Complex phenotypes captured on histological slides represent the biological processes at play in individual cancers, but the link to underlying molecular classification has not been clarified or systematised. In colorectal cancer (CRC), histological grading is a poor predictor of disease progression, and consensus molecular subtypes (CMSs) cannot be distinguished without gene expression profiling. We hypothesise that image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data and to resolve unclassifiable or heterogeneous cases. In this study, we present an image-based approach to predict CRC CMS from standard H&E sections using deep learning. Design Training and evaluation of a neural network were performed using a total of n=1206 tissue sections with comprehensive multi-omic data from three independent datasets (training on FOCUS trial, n=278 patients; test on rectal cancer biopsies, GRAMPIAN cohort, n=144 patients; and The Cancer Genome Atlas (TCGA), n=430 patients). Ground truth CMS calls were ascertained by matching random forest and single sample predictions from CMS classifier. Results Image-based CMS (imCMS) accurately classified slides in unseen datasets from TCGA (n=431 slides, AUC)=0.84) and rectal cancer biopsies (n=265 slides, AUC=0.85). imCMS spatially resolved intratumoural heterogeneity and provided secondary calls correlating with bioinformatic prediction from molecular data. imCMS classified samples previously unclassifiable by RNA expression profiling, reproduced the expected correlations with genomic and epigenetic alterations and showed similar prognostic associations as transcriptomic CMS. Conclusion This study shows that a prediction of RNA expression classifiers can be made from H&E images, opening the door to simple, cheap and reliable biological stratification within routine workflows.

Background: Outcomes in locally advanced rectal cancer are improved by neoadjuvant therapy followed by surgical resection. Some patients respond completely to preoperative treatment. Therefore, predicting the pathological response to preoperative therapy is of clinical importance. Accurate prediction would allow for tailored approaches to neoadjuvant therapy. Methods: All patients undergoing resection of rectal adenocarcinoma after neoadjuvant therapy between 2006 and 2015 were included in this cohort study. Patients were identified from a prospectively collected database and data were supplemented retrospectively with full blood count at diagnosis. Specimens resected following neoadjuvant therapy were graded according to pathological response. Follow-up data was obtained from the national registry. The primary outcome was complete pathological response. Results: Of 330 patients, 71 (21.5%) responded completely to preoperative therapy. Median age was 66 and 65% were male (n = 215). White cell count (WCC) was the most predictive marker, for predicting pCR; area under the curve (AUC) 0.666. This was higher than neutrophil/platelet ratio (AUC 0.652) or neutrophil/lymphocyte ratios (AUC = 0.437). Kaplan-Meier survival analysis showed those patients with WCC > 8 had poorer survival than those with WCC < 8 (p = 0.009). Conclusion: Routinely collected haematology samples at the point of diagnosis can assist in predicting for complete response to neoadjuvant therapy. Although novel biomarkers will have a greater predictive value, this clinically available value test could help to assist in risk stratification of patients using routinely collected laboratory tests.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B−/− mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75–87. ©2017 AACR.

Neoadjuvant chemoradiotherapy is commonly used to treat rectal cancer but patients have different levels of response and/or toxic effects. As part of the Stratification in COloRecTal cancer (S:CORT) programme, we collected 257 rectal biopsies from two cohorts: Grampian (single hospital) and Aristotle (clinical trial). All patients had been subsequently treated with identical regimen of neoadjuvant radiotherapy and capecitabine. We performed trancriptomic, mutation and copy number profiling and aimed to identify biomarkers associated with the robust pathological endpoint of complete response (CR). Key biological determinants were identified by linear regression of different pre-defined, hypothesis-driven biomarkers for radiotherapy response, adjusted by the known confounders T and N stage. A novel RNA signature was derived using a personalised bioinformatical pipeline using a wide range of machine learning approaches. Results were validated in a publicly available transcriptomic cohort of 107 patients treated with similar dose of radiotherapy and 5-fluorouracil infusion. Further comparision of the biological determinants and the novel RNA signature were performed in the same cohorts and also TCGA by linear regression. Previously published transcriptomic signatures were retrieved and assessed in the validation, unseen cohort. Grampian and Aristotle cohorts had similar statistical power and showed similar associations of CR with biological candidates, 10 of them being significant or borderline (p The immune, stromal and epithelial components of rectal tumours are important players for prediction of CR to radiotherapy in rectal cancer. A 33-gene transcriptomic biomarker can be used to effectively select patients that are highly likely to achieve CR allowing organ preservation while modulation of the relevant biological features in the other patients may be tested to improve their poor outcome with current treatment strategies. Citation Format: Enric Domingo, Sanjay Rathee, Andrew Blake, Leslie M. Samuel, Graeme I. Murray, David Sebag-Montefiore, Simon Gollins, Nicholas West, Rubina Begum, Marian Duggan, Laura White, Susan Richman, Philip Quirke, James Robineau, Keara Redmond, Aikaterini Chatzipli, Ultan McDermott, Ian Tomlinson, Philip Dunne, Francesca Buffa, Tim Maughan. Stratification of radiotherapy and fluoropyrimidine-based chemotherapy from multi-omic profiling in rectal cancer biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB129.

Purpose The Cancer Esophagus Gefitinib trial demonstrated improved progression-free survival with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib relative to placebo in patients with advanced esophageal cancer who had disease progression after chemotherapy. Rapid and durable responses were observed in a minority of patients. We hypothesized that genetic aberration of the EGFR pathway would identify patients benefitting from gefitinib. Methods A prespecified, blinded molecular analysis of Cancer Esophagus Gefitinib trial tumors was conducted to compare efficacy of gefitinib with that of placebo according to EGFR copy number gain (CNG) and EGFR, KRAS, BRAF, and PIK3CA mutation status. EGFR CNG was determined by fluorescent in situ hybridization (FISH) using prespecified criteria and EGFR FISH-positive status was defined as high polysomy or amplification. Results Biomarker data were available for 340 patients. In EGFR FISH-positive tumors (20.2%), overall survival was improved with gefitinib compared with placebo (hazard ratio [HR] for death, 0.59; 95% CI, 0.35 to 1.00; P = .05). In EGFR FISH-negative tumors, there was no difference in overall survival with gefitinib compared with placebo (HR for death, 0.90; 95% CI, 0.69 to 1.18; P = .46). Patients with EGFR amplification (7.2%) gained greatest benefit from gefitinib (HR for death, 0.21; 95% CI, 0.07 to 0.64; P = .006). There was no difference in overall survival for gefitinib versus placebo for patients with EGFR, KRAS, BRAF, and PIK3CA mutations, or for any mutation versus none. Conclusion EGFR CNG assessed by FISH appears to identify a subgroup of patients with esophageal cancer who may benefit from gefitinib as a second-line treatment. Results of this study suggest that anti-EGFR therapies should be investigated in prospective clinical trials in different settings in EGFR FISH-positive and, in particular, EGFR-amplified esophageal cancer.

BACKGROUNDSignet ring cell colorectal cancer (SRCCa) has a bleak prognosis. Employing molecular pathology techniques we investigated the potential of precision medicine in this disease.METHODSUsing test (n=26) and validation (n=18) cohorts, analysis of mutations, DNA methylation and transcriptome was carried out. Microsatellite instability (MSI) status was established and immunohistochemistry (IHC) was used to test for adaptive immunity (CD3) and the immune checkpoint PDL1.RESULTS DNA methylation data split the cohorts into hypermethylated (n=18, 41%) and hypomethylated groups (n=26, 59%). The hypermethylated group predominant in the proximal colon was enriched for CpG island methylator phenotype (CIMP), BRAF V600E mutation and MSI (PC) across both groups. No statistically significant difference in outcome was observed between the two groups.CONCLUSIONS Our data show that SRCCa phenotype comprises two distinct genotypes. The MSI(+)/CIMP(+)/BRAF V600E(+)/CD3(+)/PDL1(+) hypermethylated genotype is an ideal candidate for immune checkpoint inhibitor therapy. In addition, one fourth of SRCCa cases can potentially be targeted by KIT inhibitors.

Background: Colorectal cancer is a common malignancy and one of the leading causes of cancer-related deaths. The metabolism of omega fatty acids has been implicated in tumour growth and metastasis. Methods: This study has characterised the expression of omega fatty acid metabolising enzymes CYP4A11, CYP4F11, CYP4V2 and CYP4Z1 using monoclonal antibodies we have developed. Immunohistochemistry was performed on a tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosa. Results: The differential expression of CYP4A11 and CYP4F11 showed a strong association with survival in both the whole patient cohort (hazard ratio (HR)=1.203, 95% CI=1.092–1.324, χ2=14.968, P=0.001) and in mismatch repair-proficient tumours (HR=1.276, 95% CI=1.095–1.488, χ2=9.988, P=0.007). Multivariate analysis revealed that the differential expression of CYP4A11 and CYP4F11 was independently prognostic in both the whole patient cohort (P=0.019) and in mismatch repair proficient tumours (P=0.046). Conclusions: A significant and independent association has been identified between overall survival and the differential expression of CYP4A11 and CYP4F11 in the whole patient cohort and in mismatch repair-proficient tumours.

Background Immunohistochemical quantification of the immune response is prognostic for colorectal cancer (CRC). Here, we evaluate the suitability of alternative immune classifiers on prognosis and assess whether they relate to biological features amenable to targeted therapy. Methods Overall survival by immune (CD3, CD4, CD8, CD20 and FOXP3) and immune-checkpoint (ICOS, IDO-1 and PD-L1) biomarkers in independent CRC cohorts was evaluated. Matched mutational and transcriptomic data were interrogated to identify associated biology. Results Determination of immune-cold tumours by combined low-density cell counts of CD3, CD4 and CD8 immunohistochemistry constituted the best prognosticator across stage II–IV CRC, particularly in patients with stage IV disease (HR 1.98 [95% CI: 1.47–2.67]). These immune-cold CRCs were associated with tumour hypoxia, confirmed using CAIX immunohistochemistry (P = 0.0009), which may mediate disease progression through common biology (KRAS mutations, CRIS-B subtype and SPP1 mRNA overexpression). Conclusions Given the significantly poorer survival of immune-cold CRC patients, these data illustrate that assessment of CD4-expressing cells complements low CD3 and CD8 immunohistochemical quantification in the tumour bulk, potentially facilitating immunophenotyping of patient biopsies to predict prognosis. In addition, we found immune-cold CRCs to associate with a difficult-to-treat, poor prognosis hypoxia signature, indicating that these patients may benefit from hypoxia-targeting clinical trials.

Cancer comprises a collection of highly proliferative and heterogeneous cells growing within an adaptive and evolving tumour microenvironment. Cancer survival rates have significantly improved following decades of cancer research. However, many experimental and preclinical studies do not translate to the bedside, reflecting the challenges of modelling the complexities and multicellular basis of human disease. Organoids are novel, complex, three-dimensional ex vivo tissue cultures that are derived from embryonic stem cells, induced pluripotent stem cells or tissue-resident progenitor cells, and represent a near-physiological model for studying cancer. Organoids develop by self-organisation, and can accurately represent the diverse genetic, cellular and pathophysiological hallmarks of cancer. In addition, co-culture methods and the ability to genetically manipulate these organoids have widened their utility in cancer research. Organoids thus offer a new and exciting platform for studying cancer and directing personalised therapies. This review aims to highlight how organoids are shaping the future of cancer research.

Objective Complex phenotypes captured on histological slides represent the biological processes at play in individual cancers, but the link to underlying molecular classification has not been clarified or systematised. In colorectal cancer (CRC), histological grading is a poor predictor of disease progression, and consensus molecular subtypes (CMSs) cannot be distinguished without gene expression profiling. We hypothesise that image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data and to resolve unclassifiable or heterogeneous cases. In this study, we present an image-based approach to predict CRC CMS from standard H&E sections using deep learning. Design Training and evaluation of a neural network were performed using a total of n=1206 tissue sections with comprehensive multi-omic data from three independent datasets (training on FOCUS trial, n=278 patients; test on rectal cancer biopsies, GRAMPIAN cohort, n=144 patients; and The Cancer Genome Atlas (TCGA), n=430 patients). Ground truth CMS calls were ascertained by matching random forest and single sample predictions from CMS classifier. Results Image-based CMS (imCMS) accurately classified slides in unseen datasets from TCGA (n=431 slides, AUC)=0.84) and rectal cancer biopsies (n=265 slides, AUC=0.85). imCMS spatially resolved intratumoural heterogeneity and provided secondary calls correlating with bioinformatic prediction from molecular data. imCMS classified samples previously unclassifiable by RNA expression profiling, reproduced the expected correlations with genomic and epigenetic alterations and showed similar prognostic associations as transcriptomic CMS. Conclusion This study shows that a prediction of RNA expression classifiers can be made from H&E images, opening the door to simple, cheap and reliable biological stratification within routine workflows.

The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene‐negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA‐mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage‐independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC–Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T‐box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

// Rebecca Swan 1 , Abdo Alnabulsi 1, 2 , Beatriz Cash 2 , Ayham Alnabulsi 2 , Graeme I. Murray 1 1 Pathology, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, AB25, 2ZD, UK 2 Vertebrate Antibodies Ltd, Zoology Department, University of Aberdeen, Aberdeen, AB24 2TZ, UK Correspondence to: Graeme I. Murray, email: g.i.murray@abdn.ac.uk Keywords: biomarker, colorectal cancer, cytochrome P450, oxysterol, prognosis Received: February 15, 2016 Accepted: May 26, 2016 Published: June 22, 2016 ABSTRACT Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis. Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis. Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (χ 2 =12.984, p

Upper gastrointestinal cancers originating in the oesophagus and stomach often present late and have a very poor prognosis. Treatment options include surgery for localised disease but, increasingly, neoadjuvant chemotherapy and radiotherapy are being employed to improve outcome. There is often a variable response to neoadjuvant treatment between individual patients and side effects are relatively common. There is an urgent need for novel biomarkers of upper gastrointestinal cancer, not only to improve screening and early diagnosis of the oesophageal and gastric cancers when treatment options are potentially more effective, but also to accurately guide therapy in more advanced disease. The development of predictive biomarkers will also help to more effectively identify those patients that will benefit from targeted therapies. Although many promising results have been derived from these studies there remains a lack of validated clinically applicable biomarkers available for translation into routine clinical use. This review will provide an overview of the recent proteomic research on upper gastrointestinal cancer protein biomarker identification and validation. The challenges faced in the development of validated, clinically acceptable and accurate protein biomarkers will also be discussed, along with possible areas of future progress.

This review aims to provide an overview of recent developments regarding the roles of MMPs in tumour invasion and metastasis. Much of the mortality burden belonging to cancer relates to its ability to invade adjacent tissue and form metastases at distant sites. This would not be possible without remodelling of the ECM, a process which is enabled by the functions of MMPs. Recent studies provide a better understanding of the importance of the biophysical nature of the ECM, how this influences cancer cell motility, and how MMPs act to modify matrix stiffness. The regulation of MMPs and the role of immune cell generated MMPs has also become better understood. All of this provides a framework for the therapeutic targeting of MMPs and recent advances in the development of selective MMPs inhibitors are also reviewed. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction Cathelicidin (LL-37) is an innate anti-microbial peptide. Previous in vitro studies suggest a protective role in colonic carcinogenesis. Our recent pre-clinical data revealed that genetic knock out of cathelicidin led to increased size and number of colorectal tumours in the azoxymethane mediated murine model of colorectal cancer (CRC). Cathelicidin expression has not been characterised in human colorectal cancer. Methods Intensity of epithelial cytoplasmic LL-37 was assessed immunohistochemically in a tissue microarray representing 650 colorectal cancers and 50 paired normal colorectal mucosa samples. Tissue was obtained from chemotherapy and radiotherapy naive patients obtained at time of surgery for primary colonic cancer, sourced from the Grampian Tissue Biorepository. Ethical approval was granted by the Grampian Biorepository Scientific Access Group. Clinico-pathological data were available for each case, including survival up to 18.2 years post-resection. Expression intensity of LL-37 was independently assessed by two observers, blind to clinico-pathological data, as absent, weak, moderate or strong. Descriptive analysis, χ2 test, Fisher’s exact test and log-rank survival analysis was performed using IBM SPSS Statistics (Version 24.0.0.0). Results The expression of cytoplasmic LL-37 was weaker in colorectal cancer compared to normal colonic epithelium (p Conclusions Loss of epithelial cytoplasmic LL-37 is associated with progression of colorectal cancer, confirming translation of pre-clinical data to human disease. There may be a global field change in LL-37 expression in distant non-malignant cells as CRC progresses. The functional impact of this warrants further investigation and may reveal new insight into the pathogenesis of colorectal cancer.

Introduction High Mobility Group Box-1 (HMGB1) is a ubiquitous nuclear protein that regulates gene expression. When phosphorylated, it translocates to the cytoplasm and extracellular space to impact immune responses and epithelial cell behaviour. Our previous data revealed that dynamic subcellular localisation of HMGB1 is associated with colorectal neoplastic progression, with cytoplasmic HMGB1 expression a feature of early carcinogenesis. The leading edge of cancer invasion in polyp cancers (CaP) had strong expression in both nuclear and cytoplasmic compartments. Our aim was to define the biological impact of this expression profile. Methods CD4+ helper T cells, CD8+ cytotoxic T cells, FOXP3+ regulatory T cells, CD20+ B cells and CD68+ macrophages were assessed by immunohistochemistry on endoscopically retrieved paraffin embedded CaP lesions sourced from the Grampian Tissue Biorepository (n=25). Ethical approval was granted by the Grampian Biorepository Scientific Access Group. The invasive cancer margin was identified by a gastrointestinal pathologist. The immune cell infiltrate was expressed as number of positive cells per high power field (x20) in the adjacent stroma alongside intensity of nuclear and cytoplasmic HMGB1 expression. The impact of HMGB1 on colonic epithelial cell behaviour was assessed by stimulation of Caco-2 cells (ATCC HTB-37TM) for 48–72 hours with 0, 50 and 100 ng/ml recombinant HMGB1 with (1) analysis of wound healing by scratch assay (images captured at time 0 and 24 hour intervals and analysed with ImageJ), (2) proliferation by incorporation of tritiated thymidine and (3) expression of permeability genes (CLDN2, CLDN4, OCLN, CDH1, TJP1) using TaqMan RT-qPCR, normalised to expression of GAPDH and B2M and analysed by the Livak method. Results A robust inflammatory infiltrate was identified adjacent to the invasive cancer margin with a preponderance of CD4+ T cells and CD68+ macrophages. The subcellular localisation of HMGB1 (nuclear versus cytoplasmic) did not result in phenotypically distinct populations. There was no difference in epithelial restitution in response to HMGB1 over 48 hours, although high concentration at 72 hours was associated with reduced wound healing (p Conclusions Cytoplasmic HMGB1 expression did not impact the phenotype of adjacent immune cell infiltrate at the cancer margin. HMGB1 influences growth dynamics of colonic epithelial cells and reduces the expression of the pore closing tight junction gene, CLDN4. Understanding HMGB1 driven biology in CaP could identify an important mechanism for early carcinogenesis.

Introduction There is a clinical need for new and safer treatment options for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an endogenous immunosuppressive cytokine through inhibition of Th2 and Th17 T cell responses and promotion of IL-10 secreting Tr1 T regulatory cells. Oral IL-27 clinically attenuates disease in several T cell and innate cell driven pre-clinical models of IBD. We need to translate this pre-clinical data to human IBD to underpin proof of concept for IL-27 as a new therapeutic for IBD. Our aim was to define IL-27 evoked responses in the human colonic epithelial barrier. Methods A human colon derived epithelial organoid model was established from fresh colon tissue obtained through the Grampian Tissue Biorepository. Ethical approval was granted by the Biorepository Scientific Access Group. Isolated colonic crypts were grown in a 3D matrix with conditioned media and growth factors to maturity. RT-qPCR using TaqMan probes characterised organoid permeability gene expression (CLDN2, CLDN4, OCLN, CDH1, TJP1, ECM1) normalised to GAPDH and B2M following a 48 hour stimulation with recombinant IL-27 (rIL-27) (0, 50, 100 ng/ml). Data was analysed with the Livak method and one-way ANOVA. The impact of rIL-27 on colonic epithelial growth dynamics was established on Caco-2 and HT-29 cell lines, stimulated with 0, 50 and 100 ng/ml rIL-27, defined by (1) wound scratch assays over 72 hours with digital images of wound areas captured at 24 hour intervals and analysed with ImageJ software, and (2) tritiated thymidine incorporation proliferation assays stimulated for 48 hours. Results rIL-27 evoked a differential epithelial permeability gene expression in human colonic organoids, with reduced expression of CLDN2 (p Conclusion Our data demonstrates that IL-27 enhances epithelial barrier wound healing. Gene expression data suggests that cell-cell adhesion is enhanced through increased E-cadherin expression, with a reduction in permeability through decreased expression of claudin-2 (pore forming) and increase in claudin-4 (pore closing). Tight junction function is enhanced through increased expression of occludin and tight junctional protein-1. Further studies will define the IL-27 driven permeability related protein expression profile and impact on functional permeability in organoids and whether IL-27 is a potential new treatment for IBD.

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1β inhibition with AKT activation in order to suppress GSK3β-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival. Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632–47. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517

Part I. Introduction An Introduction to Laser-Based Tissue Microdissection Techniques Stephanie Curran and Graeme I. Murray Part II. Microdissection and DNA Analysis Methacarn Fixation for Genomic DNA Analysis in Microdissected Cells Makoto Shibutani and Chikako Uneyama Multiplex Quantitative Real-Time PCR of Laser Microdissected Tissue Patrick H. Rooney Comparative Genomic Hybridization Using DNA From Laser Capture Microdissected Tissue Grace Callagy, Lucy Jackson, and Carlos Caldas Detection of Ki-ras and p53 Mutations by Laser Capture Microdissection/PCR/SSCP Deborah Dillon, Karl Zheng, Brina Negin, and Jose Costa Whole-Genome Allelotyping Using Laser Microdissected Tissue Colleen M. Feltmate and Samuel C. Mok Microdissection for Detecting Genetic Aberrations in Early and Advanced Human Urinary Bladder Cancer Arndt Hartmann, Robert Stoehr, Peter J. Wild, Wolfgang Dietmaier, and Ruth Knuechel Laser Microdissection for Microsatellite Analysis in Colon and Breast Cancer Peter J. Wild, Robert Stoehr, Ruth Knuechel, Arndt Hartmann, and Wolfgang Dietmaier Assessment of RET/PTC Oncogene Activation in Thyroid Nodules Utilizing Laser Microdissection Followed by Nested RT-PCR Giovanni Tallini and Guilherme Brandao Combined Laser-Assisted Microdissection and Short Tandem Repeat Analysis for Detection of In Situ Microchimerism After Solid Organ Transplantation Ulrich Lehmann, Anne Versmold, and Hans Kreipe Part III. RNA and Gene Expression Studies Using Microdissected Cells, Laser-Assisted Microdissection of Membrane-Mounted Tissue Sections Lise Mette Gjerdrum and Stephen Hamilton-Dutoit. Laser-Assisted Microdissection of Membrane-Mounted Sections Following Immunohistochemistry and In Situ Hybridization Lise Mette Gjerdrum and Stephen Hamilton-Dutoit Laser-Assisted Cell Microdissection Using the PALM System Patrick Micke, Arne A-stman, Joakim Lundeberg, and Fredrik Ponten Laser Microdissection and RNA Analysis Ludger Fink and Rainer Maria Bohle Gene Expression Profiling of Primary Tumor Cell Populations Using Laser Capture Microdissection, RNA Transcript Amplification, and GeneChip(R) Microarrays Veronica I. Luzzi, Victoria Holtschlag, and Mark A. Watson Quantification of Gene Expression in Mouse and Human Renal Proximal Tubules Jun-ya Kaimori, Masaru Takenaka, and Kousaku Okubo Laser Capture Microdissection for Analysis of Macrophage Gene Expression From Atherosclerotic Lesions Eugene Trogan and Edward A. Fisher Analysis of Pituitary Cells by Laser Capture Microdissection Ricardo V. Lloyd, Xiang Qian, Long Jin, Katharina Ruebel, Jill Bayliss, Shuya Zhang, and Ikuo Kobayaski Part IV. Microdissection Techniques and Applications in Proteomics Laser Capture Microdissection and Colorectal Cancer Proteomics Laura C. Lawrie and Stephanie Curran Proteomic Analysis of Human Bladder Tissue Using SELDI(R) Approach Following Microdissection Techniques Rene C. Krieg, Nadine T. Gaisa, Cloud P. Paweletz, and Ruth Knuechel Part V. Microdissection and Molecular Analysis of Microorganisms Genetic Analysis of HIV by In Situ PCR-Directed Laser Capture Microscopy of Infected Cells Daniele Marras Use of Laser Capture Microdissection Together With In Situ Hybridization and Real-Time PCR to Study the Distribution of Latent Herpes Simplex Virus Genomes in Mouse Trigeminal Ganglion Xiao-Ping Chen, Marina Mata, and David J. Fink Laser Capture Microdissection and PCR for Analysis of Human Papilloma Virus Infection Kheng Chew, Patrick H. Rooney, Margaret E. Cruickshank, and Graeme I. Murray Laser Capture Microdissection of Hepatic Stages of the Human Parasite Plasmodium falciparum for Molecular Analysis Jean-Philippe Semblat, Olivier Silvie, Jean-Francois Franetich, and Dominique Mazier Index

Objectives The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. Methods Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). Results Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. Conclusions These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection.