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Background: Tumour immunoprofiling captures tumour-microenvironment interactions and enables precision medicine. Multiplex immunofluorescence (mIF) imaging can provide comprehensive quantitative and spatial information for multiple immune markers. However, application at scale to clinical trial samples sourced from multiple institutions is challenging due to pre-analytical heterogeneity. Methods: We analysed 12'592 tissue microarray (TMA) spots from 3'545 colorectal cancers (CRC) sourced from more than 240 institutions in two clinical trials (QUASAR 2 and SCOT) stained for CD4, CD8, CD20, CD68, FoxP3, pan-cytokeratin and DAPI by mIF. TMA cores were multi-spectrally imaged by digital pathology and analysed by cell-based and pixel-based marker analysis. We developed and validated an adaptive thresholding method to account for inter- and intra-slide intensity variation in TMA analysis, compared the numerical results between analysis approaches and validated against the current gold standard of single-plex chromogenic immunohistochemistry (IHC). Results: Adaptive thresholding at a slide and spot level effectively ameliorated inter- and intra-slide intensity variation enabling high-throughput analysis of mIF-stained TMA cores by digital pathology and improving the image analysis results compared to methods using a single global threshold. Comparison of our mIF approach with CD8 IHC data derived from subsequent sections indicates the validity of our method (Spearman's rank correlation coefficients (SCC) between 0.63 and 0.66, p-value << 0.01). Evaluation of correlation between cell-based and pixel-based analysis results confirms equivalency (SCC > 0.8, p<< 0.01, except for CD20 in epithelium region) of both analytical approaches for precision immunoprofiling by mIF. Conclusions: This study reports an analytical approach to the largest multiparameter immunoprofiling study of clinical trial samples to date and establishes an adaptive thresholding method to account for inter- and intra-slide variation introduced by pre-analytical heterogeneity. This approach can enable the application of mIF immunoprofiling of clinical trial TMA datasets at scale.

Supplementary Methods; Supplementary Figures S1-S5

Supplementary Results

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary data to accompany manuscript

Purpose: Oncogene mutations contribute to colorectal cancer development. We searched for differences in oncogene mutation profiles between colorectal cancer metastases from different sites and evaluated these as markers for site of relapse.Experimental Design: One hundred colorectal cancer metastases were screened for mutations in 19 oncogenes, and further 61 metastases and 87 matched primary cancers were analyzed for genes with identified mutations. Mutation prevalence was compared between (a) metastases from liver (n = 65), lung (n = 50), and brain (n = 46), (b) metastases and matched primary cancers, and (c) metastases and an independent cohort of primary cancers (n = 604). Mutations differing between metastasis sites were evaluated as markers for site of relapse in 859 patients from the VICTOR trial.Results: In colorectal cancer metastases, mutations were detected in 4 of 19 oncogenes: BRAF (3.1%), KRAS (48.4%), NRAS (6.2%), and PIK3CA (16.1%). KRAS mutation prevalence was significantly higher in lung (62.0%) and brain (56.5%) than in liver metastases (32.3%; P = 0.003). Mutation status was highly concordant between primary cancer and metastasis from the same individual. Compared with independent primary cancers, KRAS mutations were more common in lung and brain metastases (P < 0.005), but similar in liver metastases. Correspondingly, KRAS mutation was associated with lung relapse (HR = 2.1; 95% CI, 1.2 to 3.5, P = 0.007) but not liver relapse in patients from the VICTOR trial.Conclusions: KRAS mutation seems to be associated with metastasis in specific sites, lung and brain, in colorectal cancer patients. Our data highlight the potential of somatic mutations for informing surveillance strategies. Clin Cancer Res; 17(5); 1122–30. ©2011 AACR.

Purpose:Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling.Experimental Design:Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets.Results:Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment.Conclusions:Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

Supplementary Tables S1-S4.

Figures S1-S9, Tables S1, S2

Supplementary Table from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Purpose:The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer.Experimental Design:Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial (n = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial (n = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer.Results:Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer.Conclusions:DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies.Significance:These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

Purpose:The cytokine IL22 promotes tumor progression in murine models of colorectal cancer. However, the clinical significance of IL22 in human colorectal cancer remains unclear. We sought to determine whether the IL22 pathway is associated with prognosis in human colorectal cancer, and to identify mechanisms by which IL22 can influence disease progression.Experimental Design:Transcriptomic data from stage II/III colon cancers in independent discovery (GSE39582 population-based cohort, N = 566) and verification (PETACC3 clinical trial, N = 752) datasets were used to investigate the association between IL22 receptor expression (encoded by the genes IL22RA1 and IL10RB), tumor mutation status, and clinical outcome using Cox proportional hazard models. Functional interactions between IL22 and mutant KRAS were elucidated using human colorectal cancer cell lines and primary tumor organoids.Results:Transcriptomic analysis revealed a poor-prognosis subset of tumors characterized by high expression of IL22RA1, the alpha subunit of the heterodimeric IL22 receptor, and KRAS mutation [relapse-free survival (RFS): HR = 2.93, P = 0.0006; overall survival (OS): HR = 2.45, P = 0.0023]. KRAS mutations showed a similar interaction with IL10RB and conferred the worst prognosis in tumors with high expression of both IL22RA1 and IL10RB (RFS: HR = 3.81, P = 0.0036; OS: HR = 3.90, P = 0.0050). Analysis of human colorectal cancer cell lines and primary tumor organoids, including an isogenic cell line pair that differed only in KRAS mutation status, showed that IL22 and mutant KRAS cooperatively enhance cancer cell proliferation, in part through augmentation of the Myc pathway.Conclusions:Interactions between KRAS and IL22 signaling may underlie a previously unrecognized subset of clinically aggressive colorectal cancer that could benefit from therapeutic modulation of the IL22 pathway.

supplementary methods

Supplementary Table 1-3 from Mechanisms of Inactivation of the Receptor Tyrosine Kinase EPHB2 in Colorectal Tumors

The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/β-catenin. Premalignant lesions of the colon express high levels of EPHB2 but the expression of this kinase is reduced or lost in most colorectal carcinomas. In addition, inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutation in the colon and rectum. In this study, we investigated the molecular mechanisms responsible for the inactivation of EPHB2 in colorectal tumors. We show here the presence of mutations in repetitive sequences in exon 17 of EPHB2 in 6 of 29 adenomas with microsatellite instability (MSI), and 101 of 246 MSI carcinomas (21% and 41%, respectively). Moreover, we found EPHB2 promoter hypermethylation in 54 of the 101 colorectal tumors studied (53%). Importantly, EPHB2 expression was restored after treatment of EPHB2-methylated colon cancer cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine. In conclusion, in this study, we elucidate the molecular mechanisms of inactivation of EPHB2 and show for the first time the high incidence of frameshift mutations in MSI colorectal tumors and aberrant methylation of the regulatory sequences of this important tumor suppressor gene.

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-κB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.

Personalized treatment approaches for patients with limited liver metastases from colorectal cancer are critically needed. By leveraging three large, independent cohorts of patients with colorectal liver metastases (n = 336), we found that a proliferative subtype associated with elevated CIN70 scores is linked to immune exclusion, increased metastatic proclivity, and inferior overall survival in colorectal liver metastases; however, high CIN70 scores generate a therapeutic vulnerability to DNA-damaging therapies leading to improved treatment responses. We propose CIN70 as a candidate biomarker to personalize systemic treatment options for patients with metastatic colorectal cancer. These findings are potentially broadly applicable to other human cancers.

Pathogenic germline exonuclease domain (ED) variants of POLE and POLD1 cause the Mendelian dominant condition polymerase proof-reading associated polyposis (PPAP). We aimed to describe the clinical features of all PPAP patients with probably pathogenic variants. We identified patients with a variants mapping to the EDs of POLE or POLD1 from cancer genetics clinics, a colorectal cancer (CRC) clinical trial, and systematic review of the literature. We used multiple evidence sources to separate ED variants into those with strong evidence of pathogenicity and those of uncertain importance. We performed quantitative analysis of the risk of CRC, colorectal adenomas, endometrial cancer or any cancer in the former group. 132 individuals carried a probably pathogenic ED variant (105 POLE, 27 POLD1). The earliest malignancy was colorectal cancer at 14. The most common tumour types were colorectal, followed by endometrial in POLD1 heterozygotes and duodenal in POLE heterozygotes. POLD1-mutant cases were at a significantly higher risk of endometrial cancer than POLE heterozygotes. Five individuals with a POLE pathogenic variant, but none with a POLD1 pathogenic variant, developed ovarian cancer. Nine patients with POLE pathogenic variants and one with a POLD1 pathogenic variant developed brain tumours. Our data provide important evidence for PPAP management. Colonoscopic surveillance is recommended from age 14 and upper-gastrointestinal surveillance from age 25. The management of other tumour risks remains uncertain, but surveillance should be considered. In the absence of strong genotype–phenotype associations, these recommendations should apply to all PPAP patients.

Purpose: Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. Experimental Design: Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Results: Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. Conclusions: Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

incomplete strut coverage determines the risk of stent thrombosis in the first months after stent implantation.To evaluate the potential better early healing of a novel probucol coated polymer free ultra-thin strut sirolimus eluting stent (PF-SES). [Clinical trial unique identifier: NCT02785237].Patients with two (angiographically similar) lesions with clinical indication for PCI were enrolled. The investigated stent was compared to a thin strut, bioresorbable polymer, sirolimus eluting stent (BP-SES). Every patient received both stents, one in each lesion, assigned in a randomized sequence. OCT was systematically performed at 3 months. Primary end point was the difference in the proportion of covered struts at 3 months (defined as ≥20 μm of tissue coverage). Secondary end points included differences in percentage of uncovered struts (0 μm coverage), mean strut coverage thickness, and malapposed struts' coverage proportion. Major adverse cardiac events (cardiac death, myocardial infarction, target lesion revascularization, and definite or probable stent thrombosis) at 12 months were also evaluated.70 patients were included. At 3 months, a consistent and significantly higher strut coverage rate (≥20 μm) was observed in PF-SES as compared to BP-SES, both for well apposed (87.3% versus 79.1%, p 0.001) and malapposed struts (50.4% vs 37.8%, p 0.00). Uncoverage rate (0 μm) was also significantly lower for the PF-SES (3.1% vs 5.3%, p 0.001). There were no differences in clinical endpoints.The probucol coated non-polymeric ultra-thin strut sirolimus eluting stent showed a significantly better early strut coverage at 3 months.

The genomic drivers of immune exclusion in colorectal cancer liver metastases (CRCLM) remain poorly understood. Chromosomal instability (CIN), resulting in aneuploidy and genomic rearrangements, is the central pathway of mismatch repair-proficient colorectal cancer pathogenesis; however, it is unknown whether CIN impacts the outcomes of patients with limited spread of CRCLM treated with curative intent cytotoxic chemotherapy and surgery. Herein, we examined the relationship between CIN and the molecular subtypes of CRCLM, immune signaling, treatment sensitivity, and patient outcomes in three independent CRCLM patient cohorts. We established that a previously developed 70-gene CIN signature (CIN70) is a reliable measure of CIN, encompassing features of both aneuploidy and cellular proliferation. We demonstrated that tumors with the canonical subtype of CRCLM exhibit elevated levels of CIN and aneuploidy. Genomically unstable tumors were associated with an immune-depleted tumor microenvironment, and patients with genomically unstable tumors were at increased risk for disease progression in adverse metastatic sites, resulting in poor progression-free and overall survival. However, high-CIN tumors were particularly susceptible to DNA-damaging chemotherapies, including topoisomerase inhibitors, as well as radiation therapy. Treatment with genotoxic agents depleted CIN-rich cell populations, which resulted in a concomitant increase in intratumoral CD8+ T-cells in patients with primary rectal, breast, and bladder cancer. Taken together, we propose a mechanistic explanation for why cytotoxic chemotherapy can augment anti-tumor immunity and improve outcomes in patients with genomically unstable cancers.

Outcomes inPatients with newly diagnosed mCRC were registered into FOCUS4 and tested forFOCUS4-C was conducted between April 2017 and Mar 2020 during which time 718 patients were registered; 247 (34%) wereIn this phase II randomized trial, adavosertib improved PFS compared with AM and demonstrates potential as a well-tolerated therapy for

3540 Background: The relevance of individual RAS (KRAS and NRAS) and TP53 mutations in CRC is well described, but the impact of the combination of both mutations together is less understood. RAS/TP53-mutant (mut) patients (pts) were selected in FOCUS 4-C for treatment with Adavosertib (Wee1 inhibitor) due to hypothesised sensitivity due to replication stress and loss of cell cycle checkpoint control. Initial analyses in CRC suggest that RAS/TP53-mut pts may be prognostically distinct from either mutation alone. Here we examine the impact of RAS/TP53-mut status in both early stage and metastatic CRC (mCRC). Methods: RAS and TP53 mutational status were assessed by whole gene targeted NGS or PCR in hotspot regions. Pts with RAS/TP53 status in the following studies were included after exclusion of BRAF-V600E mutants and MSI-H cases: for early-stage, QUASAR2 trial (N = 408) and an Australian community cohort (N = 654); for mCRC, FOCUS (N = 373) and FOCUS4 (N = 721). Biomarker prevalence, clinical characteristics and outcome data in the RAS/TP53-mut group were compared to pts not showing dual RAS/TP53-mut. Results: The prevalence of RAS/TP53-mut was greater in mCRC compared to early CRC (43.9% vs 25.4% respectively, p < 0.001). In early-stage (II & III) cohorts combined, RAS/TP53-mut pts were more likely to be female, have a right-sided primary tumour, and involved lymph nodes. In early CRC RAS/TP53-mut pts had worse outcome: DFS HR = 1.49[1.19-1.88], p = 0.001, and OS HR = 1.48[1.16-1.89], p = 0.001. In FOCUS, RAS/TP53-mut mCRC pts had inferior PFS with 1st line chemotherapy than not dual RAS/TP53-mut: 6.9 vs 8.6 months (HR = 1.44[1.17-1.79], p = 0.001), and also shorter post-progression survival (HR = 1.49, p = 0.001), and overall survival (14.9 vs 18.9 mths [HR = 1.60, p < 0.0005]). Consistently, during 16 weeks of induction chemotherapy for mCRC pts in FOCUS4, 27.4% of RAS/TP53-mut pts had progressive disease, compared with 18.4% in not dual RAS/TP53-mut; PFS from study registration was reduced in RAS/TP53-mut (5.3 vs 6.1 mths;HR = 1.53[1.22-1.94],p < 0.001), but no statistically significant difference in OS (13.6 vs 17.6 mths;HR = 1.27,p = 0.23). Outcomes by each of the four biomarker groups (RAS/TP53 dual mut; RASwt/TP53mut; RASmut/TP53 wt; RAS/TP53 dual wt) will be presented but in all cases the dual mut subgroup had the worst outcomes compared to the other three groups, marginally better than BRAF-V600E CRC. Conclusions: RAS/TP53 dual mutation status provides useful and readily available prognostic information in both early and mCRC, independent of MSI and BRAF status. It is associated with increased risk of recurrence in early CRC, and a higher risk of chemotherapy resistance and inferior outcomes in mCRC. Evaluation of treatment strategies in this sizeable patient group and further understanding of the underlying mechanism of poor outcomes are urgently required.

Aims: After local excision of early rectal cancer, definitive lymph node status is not available. An alternative means for accurate assessment of recurrence risk is required to determine the most appropriate subsequent management. Currently used measures are suboptimal. We assess three measures of tumour stromal content to determine their predictive value after local excision in a well-characterised cohort of rectal cancer patients without prior radiotherapy. Methods and results: A total of 143 patients were included. Haematoxylin and eosin (H&E) sections were scanned for (i) deep neural network (DNN, a machine-learning algorithm) tumour segmentation into compartments including desmoplastic stroma and inflamed stroma; and (ii) digital assessment of tumour stromal fraction (TSR) and optical DNA ploidy analysis. 3′ mRNA sequencing was performed to obtain gene expression data from which stromal and immune scores were calculated using the ESTIMATE method. Full results were available for 139 samples and compared with disease-free survival. All three methods were prognostic. Most strongly predictive was a DNN-determined ratio of desmoplastic to inflamed stroma >5.41 (P < 0.0001). A ratio of ESTIMATE stromal to immune score 36.5% (P = 0.037). Conclusions: The DNN-determined ratio of desmoplastic to inflamed ratio is a novel and powerful predictor of disease recurrence in locally excised early rectal cancer. It can be assessed on a single H&E section, so could be applied in routine clinical practice to improve the prognostic information available to patients and clinicians to inform the decision concerning further management.

BackgroundPulmonary arterial (PA) stiffness has an essential contribution to the right ventricular (RV) failure pathogenesis. A comprehensive and multiparameter risk assessment allows predicting mortality and guiding treatment decisions in PA hypertension (PAH). We characterize PA remodeling with intravascular ultrasound (IVUS) in prevalent and stable patients with PAH according to the ESC/ERS risk table and analyze the RV-PA coupling consequences.MethodsTen control subjects and 20 prevalent PAH adult patients underwent right heart catheterization (RHC) with simultaneous IVUS study. We estimated cardiac index (CI), pulmonary vascular resistance, and compliance (PVR, PAC) by standard formulas. From IVUS and RHC data, PA diameter, wall thickness/luminal diameter ratio, and indexes of stiffness (pulsatility, compliance, distensibility, incremental elastic modulus - Einc-, and the stiffness index β) were measured. We evaluated RV-PA coupling by the ratio of tricuspid annular plane systolic excursion to systolic pulmonary arterial pressure (TAPSE/sPAP). The individual average risk was calculated by assigning a score of 1 (low-risk -LR-), 2 (intermediate-risk -IR-), and 3 (high-risk -HR-) for each of seven variables (functional class, six-minute walking test, brain natriuretic peptide, right atrial area and pressure, CI, and PA oxygen saturation) and rounding the average value to the nearest integer.ResultsAll PA segments interrogated showed increased vessel diameter, wall cross-sectional area (WCSA), and stiffness in patients with PAH compared to control subjects. 45% corresponded to LR, and 55% corresponded to IR PAH patients. The different measurements of PA stiffness showed significant correlations with TAPSE/sPAP (r = 0.6 to 0.76) in PAH patients. The IR group had higher PA stiffness and lower relative WCSA than LR patients (P < 0.05), and it is associated with a lower PAC and TAPSE/sPAP (P < 0.05).ConclusionIn prevalent PAH patients, the severity of proximal PA remodeling is related to the risk stratification and associated with PAC and RV-PA coupling impairment beyond the indirect effect of the mean PA pressure. The concomitant assessment of IVUS and hemodynamic parameters at diagnosis and follow-up of PAH patients could be a feasible and safe tool for risk stratification and treatment response of the PA vasculopathy during serial hemodynamic measurements.

Background Improved markers of prognosis are needed to stratify patients with early-stage colorectal cancer to refine selection of adjuvant therapy. The aim of the present study was to develop a biomarker of patient outcome after primary colorectal cancer resection by directly analysing scanned conventional haematoxylin and eosin stained sections using deep learning. Methods More than 12 000 000 image tiles from patients with a distinctly good or poor disease outcome from four cohorts were used to train a total of ten convolutional neural networks, purpose-built for classifying supersized heterogeneous images. A prognostic biomarker integrating the ten networks was determined using patients with a non-distinct outcome. The marker was tested on 920 patients with slides prepared in the UK, and then independently validated according to a predefined protocol in 1122 patients treated with single-agent capecitabine using slides prepared in Norway. All cohorts included only patients with resectable tumours, and a formalin-fixed, paraffin-embedded tumour tissue block available for analysis. The primary outcome was cancer-specific survival. Findings 828 patients from four cohorts had a distinct outcome and were used as a training cohort to obtain clear ground truth. 1645 patients had a non-distinct outcome and were used for tuning. The biomarker provided a hazard ratio for poor versus good prognosis of 3·84 (95% CI 2·72–5·43; p

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.

Background The relation between STEMI and air pollution (AP) is scant. We aimed to investigate the short term association between AP and the incidence of STEMI, and STEMI-related ventricular arrhythmias (VA) and mortality. Methods The study was carried out in the area of Barcelona from January 2010 to December 2011. Daily STEMI rates and incidence of STEMI-related VA and mortality were obtained prospectively. The corresponding daily levels of the main pollutants were recorded as well as the atmospheric variables. Three cohorts were defined in order to minimize exposure bias. The magnitude of association was estimated using a time-series design and was adjusted according to atmospheric variables. Results The daily rate of hospital admissions for STEMI was associated with increases in PM 2.5, PM 10, lead and NO2 concentrations. VA incidence and mortality were associated with increases in PM 2.5 and PM 10 concentrations. In the most specific cohort, BCN (Barcelona) Attended & Resident, STEMI incidence was associated with increases in PM 2.5 (1.009% per 10μg/m 3 ) and PM 10 concentrations (1.005% per 10μg/m 3 ). VA was associated with increases in PM 2.5 (1.021%) and PM 10 (1.015%) and mortality was associated with increases in PM 2.5 (1.083%) and PM 10 (1.045%). Conclusions Short-term exposure to high levels of PM 2.5 and PM 10 is associated with increased daily STEMI admissions and STEMI-related VA and mortality. Exposure to high levels of lead and NO2 is associated with increased daily STEMI admissions, and NO2 with higher mortality in STEMI patients.

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.

The role of the left ventricle in ICU patients with circulatory shock has long been considered. However, acute right ventricle (RV) dysfunction causes and aggravates many common critical diseases (acute respiratory distress syndrome, pulmonary embolism, acute myocardial infarction, and postoperative cardiac surgery). Several supportive therapies, including mechanical ventilation and fluid management, can make RV dysfunction worse, potentially exacerbating shock. We briefly review the epidemiology, pathophysiology, diagnosis, and recommendations to guide management of acute RV dysfunction in ICU patients. Our aim is to clarify the complex effects of mechanical ventilation, fluid therapy, vasoactive drug infusions, and other therapies to resuscitate the critical patient optimally.

Purpose:The cytokine IL22 promotes tumor progression in murine models of colorectal cancer. However, the clinical significance of IL22 in human colorectal cancer remains unclear. We sought to determine whether the IL22 pathway is associated with prognosis in human colorectal cancer, and to identify mechanisms by which IL22 can influence disease progression.Experimental Design:Transcriptomic data from stage II/III colon cancers in independent discovery (GSE39582 population-based cohort, N = 566) and verification (PETACC3 clinical trial, N = 752) datasets were used to investigate the association between IL22 receptor expression (encoded by the genes IL22RA1 and IL10RB), tumor mutation status, and clinical outcome using Cox proportional hazard models. Functional interactions between IL22 and mutant KRAS were elucidated using human colorectal cancer cell lines and primary tumor organoids.Results:Transcriptomic analysis revealed a poor-prognosis subset of tumors characterized by high expression of IL22RA1, the alpha subunit of the heterodimeric IL22 receptor, and KRAS mutation [relapse-free survival (RFS): HR = 2.93, P = 0.0006; overall survival (OS): HR = 2.45, P = 0.0023]. KRAS mutations showed a similar interaction with IL10RB and conferred the worst prognosis in tumors with high expression of both IL22RA1 and IL10RB (RFS: HR = 3.81, P = 0.0036; OS: HR = 3.90, P = 0.0050). Analysis of human colorectal cancer cell lines and primary tumor organoids, including an isogenic cell line pair that differed only in KRAS mutation status, showed that IL22 and mutant KRAS cooperatively enhance cancer cell proliferation, in part through augmentation of the Myc pathway.Conclusions:Interactions between KRAS and IL22 signaling may underlie a previously unrecognized subset of clinically aggressive colorectal cancer that could benefit from therapeutic modulation of the IL22 pathway.

Regression of pulmonary hypertension (PH) is often incomplete after successful left-sided valve replacement (LSVR). Proximal pulmonary arterial (PPA) wall disease can be involved in patients with persistent-PH after LSVR, affecting the right ventricular to pulmonary arterial (RV-PA) coupling. Fifteen patients underwent successful LSVR at least one year ago presenting PH by echo (> 50 mmHg). Prosthesis-patient mismatch and left ventricular dysfunction were discarded. All patients underwent hemodynamic and intravascular ultrasound (IVUS) study. We estimated PPA stiffness (elastic modulus [EM]) and the relative area wall thickness (AWT). Acute vasoreactivity was assessed by inhaled nitric oxide (iNO) testing. RV-PA coupling was estimated by the tricuspid annular plane systolic excursion to systolic pulmonary arterial pressure ratio. Patients were classified as isolated post-capillary PH (Ipc-PH; pulmonary vascular resistance [PVR] ≤ 3 WU and/or diastolic pulmonary gradient [DPG] 3 WU and DPG ≥ 7 mmHg). Both Ipc-PH and Cpc-PH showed a significant increase of EM and AWT. Despite normal PVR and DPG, Ipc-PH had a significant decrease in pulmonary arterial capacitance and RV-PA coupling impairment. Cpc-PH had worse PA stiffness and RV-PA coupling to Ipc-PH ( P

Background We report here the prognostic value of ploidy and digital tumour-stromal morphometric analyses using material from 2624 patients with early stage colorectal cancer (CRC). Patients and methods DNA content (ploidy) and stroma-tumour fraction were estimated using automated digital imaging systems and DNA was extracted from sections of formalin-fixed paraffin-embedded (FFPE) tissue for analysis of microsatellite instability. Samples were available from 1092 patients recruited to the QUASAR 2 trial and two large observational series (Gloucester, n = 954; Oslo University Hospital, n = 578). Resultant biomarkers were analysed for prognostic impact using 5-year cancer-specific survival (CSS) as the clinical end point. Results Ploidy and stroma-tumour fraction were significantly prognostic in a multivariate model adjusted for age, adjuvant treatment, and pathological T-stage in stage II patients, and the combination of ploidy and stroma-tumour fraction was found to stratify these patients into three clinically useful groups; 5-year CSS 90% versus 83% versus 73% [hazard ratio (HR) = 1.77 (95% confidence interval (95% CI): 1.13–2.77) and HR = 2.95 (95% CI: 1.73–5.03), P Conclusion A novel biomarker, combining estimates of ploidy and stroma-tumour fraction, sampled from FFPE tissue, identifies stage II CRC patients with low, intermediate or high risk of CRC disease specific death, and can reliably stratify clinically relevant patient sub-populations with differential risks of tumour recurrence and may support choice of adjuvant therapy for these individuals.

Treatment of pulmonary arterial hypertension has achieved significant progress over the past 20 years. Currently, 3 groups of drugs have proven useful for the treatment of this disease: endothelin receptor antagonist, phosphodiesterase inhibitors and prostacyclin and its analogues. It is recommended to initiate treatment with one of these drugs, the choice depending on the initial severity of patient disease and the preferences of the treating physician. When the patient does not have a satisfactory response, new drugs acting at a different pathway are most commonly added. At this time, considering referral for lung transplantation could be an alternative. Most experts recommend grouping maximum experience in what is known as expert centers. Treatment has led to better survival in these patients, but there is still a long way to cure this life-threatening disease.

With the increased use of next-generation sequencing generating large amounts of genomic data, gene expression signatures are becoming critically important tools for the interpretation of these data, and are poised to have a substantial effect on diagnosis, management, and prognosis for a number of diseases. It is becoming crucial to establish whether the expression patterns and statistical properties of sets of genes, or gene signatures, are conserved across independent datasets. Conversely, it is necessary to compare established signatures on the same dataset to better understand how they capture different clinical or biological characteristics. Here we describe how to use sigQC, a tool that enables a streamlined, systematic approach for the evaluation of previously obtained gene signatures across multiple gene expression datasets. We implemented sigQC in an R package, making it accessible to users who have knowledge of file input/output and matrix manipulation in R and a moderate grasp of core statistical principles. SigQC has been adopted in basic biology and translational studies, including, but not limited to, the evaluation of multiple gene signatures for potential clinical use as cancer biomarkers. This protocol uses a previously obtained signature for breast cancer metastasis as an example to illustrate the critical quality control steps involved in evaluating its expression, variability, and structure in breast tumor RNA-sequencing data, a different dataset from that in which the signature was originally derived. We demonstrate how the outputs created from sigQC can be used for the evaluation of gene signatures on large-scale gene expression datasets.

It is unclear why among patients with first acute myocardial infarction and an occluded culprit artery only some present ST segment elevation. In fact, there is no study that compares the angiographic area at risk and the collateral circulation in first NSTEMI vs STEMI patients.205 patients admitted for myocardial infarction with occluded culprit artery were included, 132 STEMI and 73 NSTEMI. Demographic data, the area at risk determined by the BARI score and collateral supply by the Rentrop score from the 2 groups were compared. NSTEMI patients showed lower peak Tn I than STEMI in the overall group but also in the 3 subsets with different culprit arteries (p .001). They also presented a higher rate of left circumflex coronary artery (CFX) as culprit artery (52% vs 14%, p .001), smaller BARI score area of the culprit artery (5.4 vs 7.6, p .001), and higher frequency of well-developed collaterals (Rentrop score ≥ 2, 1.82 vs 0.41, p .001). The latter was also higher in each of the 3 different culprit arteries (p = .002-0.001) Among 38 NSTEMI patients with CFX occlusion, 20 with ≥1 mm ST depression in V2 to V4 (possible posterior infarction) showed a similar Rentrop score than the 18 with other ECG changes but lower Tn I peak (p = .012).In first acute myocardial infarction with an occluded culprit artery NSTEMI patients - including those with possible posterior infarction - present smaller infarct size and higher collateral blood supply than STEMI patients in each of the 3 main culprit arteries.

With the increase in next generation sequencing generating large amounts of genomic data, gene expression signatures are becoming critically important tools, poised to make a large impact on the diagnosis, management and prognosis for a number of diseases. Increasingly, it is becoming necessary to determine whether a gene expression signature may apply to a dataset, but no standard quality control methodology exists. In this work, we introduce the first protocol, implemented in an R package sigQC, enabling a streamlined methodological and standardised approach for the quality control validation of gene signatures on independent data sets. The emphasis in this work is in showing the critical quality control steps involved in the generation of a clinically and biologically useful, transportable gene signature, including ensuring sufficient expression, variability, and autocorrelation of a signature. We demonstrate the application of the protocol in this work, showing how the outputs created from sigQC may be used for the evaluation of gene signatures on large-scale gene expression data in cancer.

Intratumoural T-cell infiltrate intensity cortes wrelaith clinical outcome in stage II/III colorectal cancer (CRC). We aimed to determine whether this association varies across this heterogeneous group.We performed a pooled analysis of 1804 CRCs from the QUASAR2 and VICTOR trials. Intratumoural CD8In QUASAR2, intratumoural CD8The prognostic value of intratumoural CD8