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1.
NPJ Precis Oncol ; 6(1): 72, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241710

RESUMO

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.

2.
J Immunother Cancer ; 10(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36137652

RESUMO

BACKGROUND AND AIMS: The role of inflammatory immune responses in colorectal cancer (CRC) development and response to therapy is a matter of intense debate. While inflammation is a known driver of CRC, inflammatory immune infiltrates are a positive prognostic factor in CRC and predispose to response to immune checkpoint blockade (ICB) therapy. Unfortunately, over 85% of CRC cases are primarily unresponsive to ICB due to the absence of an immune infiltrate, and even the cases that show an initial immune infiltration can become refractory to ICB. The identification of therapy supportive immune responses in the field has been partially hindered by the sparsity of suitable mouse models to recapitulate the human disease. In this study, we aimed to understand how the dysregulation of the complement anaphylatoxin C3a receptor (C3aR), observed in subsets of patients with CRC, affects the immune responses, the development of CRC, and response to ICB therapy. METHODS: We use a comprehensive approach encompassing analysis of publicly available human CRC datasets, inflammation-driven and newly generated spontaneous mouse models of CRC, and multiplatform high-dimensional analysis of immune responses using microbiota sequencing, RNA sequencing, and mass cytometry. RESULTS: We found that patients' regulation of the complement C3aR is associated with epigenetic modifications. Specifically, downregulation of C3ar1 in human CRC promotes a tumor microenvironment characterized by the accumulation of innate and adaptive immune cells that support antitumor immunity. In addition, in vivo studies in our newly generated mouse model revealed that the lack of C3a in the colon activates a microbiota-mediated proinflammatory program which promotes the development of tumors with an immune signature that renders them responsive to the ICB therapy. CONCLUSIONS: Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance antitumor immunity in CRC. C3aR can thus be exploited to overcome ICB resistance in a larger group of patients with CRC.


Assuntos
Neoplasias Colorretais , Inibidores de Checkpoint Imunológico , Anafilatoxinas , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação para Baixo , Humanos , Fatores Imunológicos , Imunoterapia/métodos , Inflamação/patologia , Camundongos , Microambiente Tumoral
3.
Clin Cancer Res ; 28(18): 4056-4069, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35792866

RESUMO

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.


Assuntos
Neoplasias Ovarianas , Neoplasias da Próstata , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/patologia , Células Estromais/metabolismo , Transcriptoma , Microambiente Tumoral/genética
4.
Int J Cardiol ; 360: 13-20, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35472561

RESUMO

BACKGROUND: incomplete strut coverage determines the risk of stent thrombosis in the first months after stent implantation. AIMS: 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]. METHODS: 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. RESULTS: 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. CONCLUSION: The probucol coated non-polymeric ultra-thin strut sirolimus eluting stent showed a significantly better early strut coverage at 3 months.


Assuntos
Stents Farmacológicos , Intervenção Coronária Percutânea , Implantes Absorvíveis , Humanos , Intervenção Coronária Percutânea/efeitos adversos , Polímeros , Probucol , Desenho de Prótese , Sirolimo , Stents , Tomografia de Coerência Óptica , Resultado do Tratamento
5.
Fam Cancer ; 21(2): 197-209, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-33948826

RESUMO

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.


Assuntos
Neoplasias Colorretais , Neoplasias do Endométrio , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , DNA Polimerase II/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/terapia , Feminino , Mutação em Linhagem Germinativa , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Propilaminas
6.
J Clin Oncol ; 39(33): 3705-3715, 2021 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-34538072

RESUMO

PURPOSE: Outcomes in RAS-mutant metastatic colorectal cancer (mCRC) remain poor and patients have limited therapeutic options. Adavosertib is the first small-molecule inhibitor of WEE1 kinase. We hypothesized that aberrations in DNA replication seen in mCRC with both RAS and TP53 mutations would sensitize tumors to WEE1 inhibition. METHODS: Patients with newly diagnosed mCRC were registered into FOCUS4 and tested for TP53 and RAS mutations. Those with both mutations who were stable or responding after 16 weeks of chemotherapy were randomly assigned 2:1 between adavosertib and active monitoring (AM). Adavosertib (250 mg or 300 mg) was taken orally once on days 1-5 and days 8-12 of a 3-week cycle. The primary outcome was progression-free survival (PFS), with a target hazard ratio (HR) of 0.5 and 80% power with a one-sided 0.025 significance level. RESULTS: FOCUS4-C was conducted between April 2017 and Mar 2020 during which time 718 patients were registered; 247 (34%) were RAS/TP53-mutant. Sixty-nine patients were randomly assigned from 25 UK hospitals (adavosertib = 44; AM = 25). Adavosertib was associated with a PFS improvement over AM (median 3.61 v 1.87 months; HR = 0.35; 95% CI, 0.18 to 0.68; P = .0022). Overall survival (OS) was not improved with adavosertib versus AM (median 14.0 v 12.8 months; HR = 0.92; 95% CI, 0.44 to 1.94; P = .93). In prespecified subgroup analysis, adavosertib activity was greater in left-sided tumors (HR = 0.24; 95% CI, 0.11 to 0.51), versus right-sided (HR = 1.02; 95% CI, 0.41 to 2.56; interaction P = .043). Adavosertib was well-tolerated; grade 3 toxicities were diarrhea (9%), nausea (5%), and neutropenia (7%). CONCLUSION: In this phase II randomized trial, adavosertib improved PFS compared with AM and demonstrates potential as a well-tolerated therapy for RAS/TP53-mutant mCRC. Further testing is required in this sizable population of unmet need.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Mutação , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinonas/uso terapêutico , Proteína Supressora de Tumor p53/genética , Conduta Expectante/estatística & dados numéricos , Proteínas ras/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Qualidade de Vida , Taxa de Sobrevida
7.
Histopathology ; 79(6): 947-956, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34174109

RESUMO

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 <1.19 was also predictive of disease-free survival (P = 0.00051), as was stromal fraction >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.


Assuntos
Recidiva Local de Neoplasia/patologia , Neoplasias Retais/patologia , Células Estromais/patologia , Microambiente Tumoral , Idoso , Estudos de Coortes , Feminino , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Pessoa de Meia-Idade , Redes Neurais de Computação , Neoplasias Retais/cirurgia , Estudos Retrospectivos
8.
Front Physiol ; 12: 631326, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34012405

RESUMO

BACKGROUND: Pulmonary 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. METHODS: Ten 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 ß) 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. RESULTS: All 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). CONCLUSION: In 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.

9.
Epigenomes ; 5(1): 6, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33777442

RESUMO

Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. METHODS: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. RESULTS: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. CONCLUSIONS: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types.

10.
Cancer Res ; 81(7): 1667-1680, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33558336

RESUMO

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.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Carcinogênese/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Polimorfismo de Nucleotídeo Único/fisiologia , Prognóstico , Fatores de Risco , Transdução de Sinais/genética , Resultado do Tratamento
12.
Gut ; 70(3): 544-554, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32690604

RESUMO

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.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Aprendizado Profundo , Regulação Neoplásica da Expressão Gênica/genética , RNA/genética , Biomarcadores Tumorais/genética , Biópsia , Consenso , Conjuntos de Dados como Assunto , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Gradação de Tumores , Fenótipo , Valor Preditivo dos Testes , Prognóstico
13.
Clin Cancer Res ; 27(1): 288-300, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33028592

RESUMO

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.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bioensaio/métodos , Biomarcadores Tumorais/genética , Neoplasias Colorretais/terapia , Dano ao DNA/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia Adjuvante/métodos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Dano ao DNA/efeitos dos fármacos , Análise Mutacional de DNA , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Leucovorina/farmacologia , Leucovorina/uso terapêutico , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Terapia Neoadjuvante/métodos , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Intervalo Livre de Progressão , Ensaios Clínicos Controlados Aleatórios como Assunto
14.
ESMO Open ; 5(6): e001040, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33219056

RESUMO

BACKGROUND: There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy. MATERIALS AND METHODS: RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression. RESULTS: Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028). CONCLUSIONS: Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.


Assuntos
Neoplasias Colorretais , Recidiva Local de Neoplasia , Quimioterapia Adjuvante , Proteínas Cromossômicas não Histona/uso terapêutico , Cromossomos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fatores de Troca do Nucleotídeo Guanina/uso terapêutico , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias
15.
Mol Oncol ; 14(10): 2384-2402, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32671911

RESUMO

Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs  > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.


Assuntos
Imunofluorescência/métodos , Microambiente Tumoral , Fluxo de Trabalho , Epitopos/imunologia , Humanos , Imageamento Tridimensional , Reprodutibilidade dos Testes
16.
Br J Cancer ; 123(8): 1280-1288, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32684627

RESUMO

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.


Assuntos
Neoplasias Colorretais/mortalidade , Hipóxia Tumoral/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Neoplasias Colorretais/imunologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico
17.
Nat Commun ; 11(1): 1274, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152270

RESUMO

Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.


Assuntos
Adenosina Trifosfatases/metabolismo , Adutos de DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Reparo do DNA , Replicação do DNA , Epistasia Genética , Humanos , Proteínas de Membrana/química , Diester Fosfórico Hidrolases/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação
18.
Lancet ; 395(10221): 350-360, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32007170

RESUMO

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<0·0001) in the primary analysis of the validation cohort, and 3·04 (2·07-4·47; p<0·0001) after adjusting for established prognostic markers significant in univariable analyses of the same cohort, which were pN stage, pT stage, lymphatic invasion, and venous vascular invasion. INTERPRETATION: A clinically useful prognostic marker was developed using deep learning allied to digital scanning of conventional haematoxylin and eosin stained tumour tissue sections. The assay has been extensively evaluated in large, independent patient populations, correlates with and outperforms established molecular and morphological prognostic markers, and gives consistent results across tumour and nodal stage. The biomarker stratified stage II and III patients into sufficiently distinct prognostic groups that potentially could be used to guide selection of adjuvant treatment by avoiding therapy in very low risk groups and identifying patients who would benefit from more intensive treatment regimes. FUNDING: The Research Council of Norway.


Assuntos
Neoplasias Colorretais/diagnóstico , Aprendizado Profundo , Idoso , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Detecção Precoce de Câncer/métodos , Amarelo de Eosina-(YS)/metabolismo , Feminino , Hematoxilina/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos
19.
Gut ; 69(6): 1092-1103, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31563876

RESUMO

OBJECTIVE: Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, ß-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. DESIGN: We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. RESULTS: Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). CONCLUSIONS: Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.


Assuntos
Neoplasias Colorretais/diagnóstico , Transdução de Sinais/genética , Proteína Wnt1/metabolismo , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Marcadores Genéticos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Wnt1/genética
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