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Gastrointestinal (GI) cancers are highly heterogeneous at multiple levels. Tumor heterogeneity can be captured by molecular profiling, such as genetic, epigenetic, proteomic, and transcriptomic classification. Transcriptomic subtyping has the advantage of combining genetic and epigenetic information, cancer cell-intrinsic properties, and the tumor microenvironment (TME). Unsupervised transcriptomic subtyping systems of different GI malignancies have gained interest because they reveal shared biological features across cancers and bear prognostic and predictive value. Importantly, transcriptomic subtypes accurately reflect complex phenotypic states varying not only per tumor region, but also throughout disease progression, with consequences for clinical management. Here, we discuss methodologies of transcriptomic subtyping, proposed taxonomies for GI malignancies, and the challenges posed to clinical implementation, highlighting opportunities for future transcriptomic profiling efforts to optimize clinical impact.
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Background: The occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC. Methods: We studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF). Results: The human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses. Conclusion: In conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.
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Neoplasias Colorrectales , Modelos Animales de Enfermedad , Neoplasias Peritoneales , Animales , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/inmunología , Humanos , Ratones , Masculino , Femenino , Línea Celular Tumoral , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Anciano , Microambiente Tumoral/inmunología , Células Asesinas Naturales/inmunologíaRESUMEN
The differentiation trajectories defining enteroendocrine (EE) cell heterogeneity remain obscure. In this issue of Cell Stem Cell, Singh et al.1 map the differentiation landscape of EE cells, identifying early oscillating cell progenitor states, which play a critical role in generating terminal EE cell diversity.
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Diferenciación Celular , Animales , Células Enteroendocrinas/citología , Células Enteroendocrinas/metabolismo , Humanos , Células Madre/citologíaRESUMEN
Cervical cancer is the fourth most common cancer in women worldwide and is caused by persistent infection with high-risk types of human papillomavirus (HPV). HPV viral load, the amount of HPV DNA in a sample, has been suggested to correlate with cervical disease severity, and with clinical outcome of cervical cancer. In this systematic review, we searched three databases (EMBASE, PubMed, Web of Science) to examine the current evidence on the association between HPV viral load in cervical samples and disease severity, as well as clinical outcome. After exclusion of articles not on HPV, cervical cancer, or containing clinical outcomes, 85 original studies involving 173 746 women were included. The vast majority (73/85 = 85.9%) reported that a higher viral load was correlated with higher disease severity or worse clinical outcome. Several studies reported either no correlation (3/85 = 3.5%), or the opposite correlation (9/85 = 10.6%); possible reasons being different categorization of HPV viral load levels, or the use of specific sampling methods. Despite variations in study design and populations, the above findings suggest that HPV viral load is correlated to clinical outcome, and may become an important biomarker for treatment selection and response monitoring for cervical cancer.
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Papillomaviridae , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Carga Viral , Humanos , Femenino , Infecciones por Papillomavirus/virología , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Papillomaviridae/clasificación , Neoplasias del Cuello Uterino/virología , Índice de Severidad de la Enfermedad , ADN Viral , Enfermedades del Cuello del Útero/virología , Virus del Papiloma HumanoRESUMEN
Consensus Molecular Subtype (CMS) classification of colorectal cancer (CRC) tissues is complicated by RNA degradation upon formalin-fixed paraffin-embedded (FFPE) preservation. Here, we present an FFPE-curated CMS classifier. The CMSFFPE classifier was developed using genes with a high transcript integrity in FFPE-derived RNA. We evaluated the classification accuracy in two FFPE-RNA datasets with matched fresh-frozen (FF) RNA data, and an FF-derived RNA set. An FFPE-RNA application cohort of metastatic CRC patients was established, partly treated with anti-EGFR therapy. Key characteristics per CMS were assessed. Cross-referenced with matched benchmark FF CMS calls, the CMSFFPE classifier strongly improved classification accuracy in two FFPE datasets compared with the original CMSClassifier (63.6% versus 40.9% and 83.3% versus 66.7%, respectively). We recovered CMS-specific recurrence-free survival patterns (CMS4 versus CMS2: hazard ratio 1.75, 95% CI 1.24-2.46). Key molecular and clinical associations of the CMSs were confirmed. In particular, we demonstrated the predictive value of CMS2 and CMS3 for anti-EGFR therapy response (CMS2&3: odds ratio 5.48, 95% CI 1.10-27.27). The CMSFFPE classifier is an optimized FFPE-curated research tool for CMS classification of clinical CRC samples.
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Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/clasificación , Neoplasias Colorrectales/patología , Adhesión en Parafina , Biomarcadores de Tumor/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Consenso , Fijación del Tejido/métodos , Masculino , Perfilación de la Expresión Génica/métodos , Anciano , Persona de Mediana Edad , Pronóstico , Regulación Neoplásica de la Expresión Génica , FormaldehídoRESUMEN
Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.
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Neoplasias Colorrectales , Mitocondrias , Neoplasias Peritoneales , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/terapia , Animales , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Ratones , Línea Celular Tumoral , Ratas , Femenino , Quimioterapia Intraperitoneal Hipertérmica/métodosRESUMEN
BACKGROUND: Small intestinal adenocarcinomas (SIAs) are rare. Hence, randomized controlled trials are lacking and understanding of the disease features is limited. This nationwide cohort investigates incidence, treatment and prognosis of SIA patients, to improve disease outcome. PATIENTS AND METHODS: Data of 2697 SIA patients diagnosed from January 1999 through December 2019 were retrieved from the Netherlands Cancer Registry and Pathology Archive. Incidence was calculated using the revised European Standardized Rate. The influence of patient and tumor characteristics on overall survival (OS) was studied using survival analyses. RESULTS: The age-standardized incidence rate almost doubled from 0.58 to 1.06 per 100,000 person-years, exclusively caused by an increase in duodenal adenocarcinomas. OS did not improve over time. Independent factors for a better OS were a younger age, jejunal tumors, Lynch syndrome and systemic therapy. Only 13.8% of resected patients was treated with adjuvant chemotherapy, which improved OS compared to surgery alone in stage III disease (HR 0.47 (0.35-0.61)), but not in the limited group of deficient mismatch repair (MMR) patients (n = 53, HR 0.93 (0.25-3.47)). In the first-line setting, CAPOX was associated with improved OS compared to FOLFOX (HR 0.51 (0.36-0.72)). For oligometastatic patients, a metastasectomy significantly improved OS (HR 0.54 (0.36-0.80)). CONCLUSIONS: The incidence of SIAs almost doubled in the past 20 years, with no improvement in OS. This retrospective non-randomized study suggests the use of adjuvant chemotherapy for stage III disease and first-line CAPOX for metastatic patients. For selected oligometastatic patients, a metastasectomy may be considered. MMR-status testing could aid in clinical decision-making.
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Adenocarcinoma , Neoplasias del Yeyuno , Humanos , Adenocarcinoma/terapia , Adenocarcinoma/tratamiento farmacológico , Estudios de Cohortes , Incidencia , Neoplasias del Yeyuno/terapia , Neoplasias del Yeyuno/tratamiento farmacológico , Pronóstico , Estudios RetrospectivosRESUMEN
PURPOSE: Hyperthermia is a potent sensitizer of radiation therapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiation therapy are still under debate. METHODS AND MATERIALS: We investigated the interval and sequence in vitro in cervical cancer cell lines, patient-derived organoids, and SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. RESULTS: All 3 approaches confirmed that shortening the interval between hyperthermia and radiation therapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis and 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥80 minutes) to 54% (interval <80 minutes). In vitro and in vivo results showed that the sequence of hyperthermia and radiation therapy did not affect the outcome. CONCLUSIONS: Shortening the interval between hyperthermia and radiation therapy significantly improves treatment outcomes. The sequence of hyperthermia and radiation therapy (before or after) does not seem to matter.
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Hipertermia Inducida , Neoplasias del Cuello Uterino , Humanos , Femenino , Animales , Ratones , Neoplasias del Cuello Uterino/radioterapia , Neoplasias del Cuello Uterino/patología , Hipertermia Inducida/métodos , Ratones Desnudos , Estudios Retrospectivos , Terapia CombinadaRESUMEN
The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.
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Ácidos Nucleicos Libres de Células , Neoplasias , Humanos , Ácidos Nucleicos Libres de Células/genética , Biomarcadores de Tumor/genética , Genómica , Biopsia Líquida , Curva ROCRESUMEN
Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.
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Técnicas de Cultivo de Célula , Neoplasias , Humanos , Células Clonales , Línea Celular , Técnicas de CocultivoRESUMEN
Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.
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Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Secuenciación de Nanoporos , Humanos , Ácidos Nucleicos Libres de Células/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Genómica/métodos , Análisis de Secuencia de ADN , ADN/genética , Biomarcadores de Tumor/genéticaRESUMEN
The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA's utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.
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In this issue of Cell Reports Medicine, Qin et al.1 present a comprehensive single-cell transcriptomics analysis of the tumor microenvironment of rectal cancer tumors before and after neoadjuvant chemotherapy.
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Terapia Neoadyuvante , Neoplasias del Recto , Humanos , Estadificación de Neoplasias , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/patología , Microambiente TumoralRESUMEN
BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.
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Ácidos Nucleicos Libres de Células , Neoplasias de la Próstata , Masculino , Humanos , Biopsia Líquida , Mitocondrias/genética , ADN Mitocondrial/genética , Neoplasias de la Próstata/genética , Biomarcadores de Tumor/genéticaRESUMEN
Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens.
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Neoplasias , ARN Largo no Codificante , Humanos , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas , ARN Largo no Codificante/genética , Neoplasias/diagnóstico , Neoplasias/genética , Genoma HumanoRESUMEN
BACKGROUND: The results of the Grupo Español Multidisciplinar en Cáncer Digestivo (GEMCAD)-1402 phase II randomized trial suggested that adding aflibercept to modified fluorouracil, oxaliplatin, and leucovorin (mFOLFOX6) induction, followed by chemoradiation and surgery, could increase the pathological complete response (pCR) rate in patients with high-risk, locally advanced rectal cancer. Here we update results up to 3 years of follow-up and evaluate the predictive value of consensus molecular subtypes identified with immunohistochemistry (IHC). METHODS: Patients with magnetic resonance imaging-defined T3c-d and/or T4 and/or N2 rectal adenocarcinoma in the middle or distal third were randomly assigned to mFOLFOX6 induction, with aflibercept (mF+A; n = 115) or without aflibercept (mF; n = 65), followed by capecitabine plus radiotherapy and surgery. The risk local relapse, distant metastases, disease-free survival (DFS), and overall survival (OS) were estimated at 3 years. Selected samples were classified via IHC into immune-infiltrate, epithelial, or mesenchymal subtypes. RESULTS: mF+A and mF had 3-year DFS of 75.2% (95% confidence interval [CI] = 66.1% to 82.2%) and 81.5% (95% CI = 69.8% to 89.1%), respectively; 3-year OS of 89.3% (95% CI = 82.0% to 93.8%) and 90.7% (95% CI = 80.6% to 95.7%), respectively; 3-year cumulative local relapse incidences of 5.2% (95% CI = 1.9% to 11.0%) and 6.1% (95% CI = 1.7% to 15.0%), respectively; and 3-year cumulative distant metastases rates of 17.3% (95% CI = 10.9% to 25.5%) and 16.9% (95% CI = 8.7% to 28.2%), respectively. pCRs were achieved in 27.5% (n = 22 of 80) and 0% (n = 0 of 10) of patients with epithelial and mesenchymal subtypes, respectively. CONCLUSION: Adding aflibercept to mFOLFOX6 induction was not associated with improved DFS or OS. Our findings suggested that consensus molecular subtypes identified with IHC subtypes could be predictive of pCR with this treatment.
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Terapia Neoadyuvante , Neoplasias del Recto , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recurrencia Local de Neoplasia/epidemiología , Recurrencia Local de Neoplasia/patología , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/patología , Fluorouracilo/uso terapéutico , Capecitabina/uso terapéutico , Quimioradioterapia/métodos , Recurrencia , Estadificación de NeoplasiasRESUMEN
BACKGROUND: The faecal immunochemical test (FIT) suffers from suboptimal performance and participation in colorectal cancer (CRC) screening. Urinary volatile organic compounds (VOCs) may be a useful alternative. We aimed to determine the diagnostic potential of urinary VOCs for CRC/adenomas. By relating VOCs to known pathways, we aimed to gain insight into the pathophysiology of colorectal neoplasia. METHODS: A systematic search was performed in PubMed, EMBASE and Web of Science. Original studies on urinary VOCs for CRC/adenoma detection with a control group were included. QUADAS-2 tool was used for quality assessment. Meta-analysis was performed by adopting a bivariate model for sensitivity/specificity. Fagan's nomogram estimated the performance of combined FIT-VOC. Neoplasm-associated VOCs were linked to pathways using the KEGG database. RESULTS: Sixteen studies-involving 837 CRC patients and 1618 controls-were included; 11 performed chemical identification and 7 chemical fingerprinting. In all studies, urinary VOCs discriminated CRC from controls. Pooled sensitivity and specificity for CRC based on chemical fingerprinting were 84% (95% CI 73-91%) and 70% (95% CI 63-77%), respectively. The most distinctive individual VOC was butanal (AUC 0.98). The estimated probability of having CRC following negative FIT was 0.38%, whereas 0.09% following negative FIT-VOC. Combined FIT-VOC would detect 33% more CRCs. In total 100 CRC-associated urinary VOCs were identified; particularly hydrocarbons, carboxylic acids, aldehydes/ketones and amino acids, and predominantly involved in TCA-cycle or alanine/aspartate/glutamine/glutamate/phenylalanine/tyrosine/tryptophan metabolism, which is supported by previous research on (colorectal)cancer biology. The potential of urinary VOCs to detect precancerous adenomas or gain insight into their pathophysiology appeared understudied. CONCLUSION: Urinary VOCs hold potential for non-invasive CRC screening. Multicentre validation studies are needed, especially focusing on adenoma detection. Urinary VOCs elucidate underlying pathophysiologic processes.