Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, accounting for more than 900,000 deaths a year worldwide. Microbial dysbiosis, including the presence of oral bacteria in the gut, has been linked to CRC. Some mechanisms by which specific microorganisms potentially drive tumorigenesis have been described, but there is a lack of studies elucidating whole microbiota activity in the tumor and their implication for the development of the disease. Here, the metatranscriptomic data of tumor and control tissue-associated microbiota (n = 18 pairs), as well as from subgingival sulcus (n = 15) of CRC patients, was analyzed. We confirmed that Fusobacterium nucleatum was more active in the tumor tissue than in the control gut mucosa. In addition, the activity of this species was positively correlated with other oral bacteria in the tumors, including Parvimonas micra, Peptostreptococcus stomatis, and Granulicatella adiacens, along with gut bacteria like Hungatella hathewayi, suggesting a potential relationship among them. Regarding bacterial gene expression, a change in the functional profile was observed, including a higher expression of genes associated with carbon metabolism in control in contrast to an increase of amino acid-related genes in tumor. Furthermore, genes implicated in the biosynthesis and transport of lipopolysaccharide were increased in tumors. Interestingly, a significantly higher expression in tumor than control tissue of potential virulence factors from F. nucleatum was found, supporting their relevance in niche colonization and tumorigenesis. Correlation analysis of the bacterial activity with the host transcriptional profile showed significant correlations of the Fusobacterium-Peptostreptotoccus-Hungatella cluster with human genes involved in inflammation and metastasis, confirming the association of this microbial consortium with tumor development. For the first time, the gene expression profiles of oral bacteria in the gut and the oral cavity were compared. The cluster of co-active bacteria identified in tumors was partially found in the oral samples, suggesting a stable interaction and potential synergy. Although there were thousands of differentially expressed genes between subgingival sulcus and tumor tissue, the expression of key virulence factors was not significantly different. In short, this study discovered new traits about tumor microbial-associated composition and activity and its connection with the oral composition that would be essential to unravel the translocation, colonization, and tumorigenesis of the CRC.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

The early detection of tumors is one of the key factors in increasing overall survival in cancer patients. A wide range of cancers still do not have a system of early diagnosis; therefore, the development of new non-invasive tools in this line is essential. Accordingly, the objective of our work was to develop a non-invasive screening method for the early detection of various carcinomas in plasma using a panel that combines two markers using RT-qPCR. A retrospective case-control study was conducted to develop a cancer screening test based on the detection of stromal and epithelial biomarkers (COL1A2 and KRT19) in plasma. The expression of biomarkers was evaluated using multiplex quantitative PCR applied to 47 cases with non-metastatic tumors and 13 control participants. For both biomarkers, a cut-off value was stablished using Youden’s J index through ROC curve analysis and areas under the curve (AUC) were calculated. The plasma mRNA expression level of both biomarkers was significantly higher in diseased versus healthy patients. Moreover, ROC curve analysis showed an AUC value of 0.897 for the combined model. This model also resulted in a cutoff value of 0.664, as well as a sensitivity of 83% and a specificity of 84.6%. These results suggest that the plasma expression levels of COL1A2 and KRT19 could a have potential role in detecting various types of cancer at the early stages. The combined analysis of both stromal and epithelial biomarkers would provide a non-invasive screening method that would allow us to differentiate patients with an active neoplastic process.

The current study assessed the performance of the fully automated RT-PCR-based Idylla™ GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK , ROS1 , RET , and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The Idylla™ GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK , 33 ROS1 , 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the Idylla™ GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK , 96.7%/99.0% for ROS1 , 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The Idylla™ GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK , ROS1 , RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

Tumor-derived extracellular vesicles (TD-EVs) have active roles as cancer hallmark enablers. EVs RNA of epithelial and stromal cells carry information that facilitates the communication processes that contribute to oncological progression, so the objective of this work was to validate by RT-PCR the presence of epithelial (KRT19; CEA) and stromal (COL1A2; COL11A1) markers in RNA of plasmatic EVs in healthy and diverse-malignancy patients for the development of a non-invasive cancer diagnosis system using liquid biopsy. Ten asymptomatic controls and 20 cancer patients were included in the study, and results showed that the isolated plasmatic EVs by scanning transmission electron microscopy (STEM) andBiomedical Research Institute A Coruña nanoparticle tracking analysis (NTA) contained most exosome structures with also a considerable percentage of microvesicles. No differences were found in concentration and size distribution between the two cohorts of patients, but significant gene expression in epithelial and mesenchymal markers between healthy donors and patients with active oncological disease was shown. Results of quantitative RT-PCR are solid and reliable for KRT19, COL1A2, and COL11A1, so the analysis of RNA extracted from TD-EVs could be a correct approach to develop a diagnostic tool in oncological processes.

PDF file - 197K, Supplemental Table 1. Characteristics of GIST patients for the immunohistochemistry study. Supplemental Table 2. Characteristics of GIST and STS patients studied in flow cytometry. Supplemental Table 3: Antibodies for flow cytometry and IHC. Supplemental Figure 1. Correlation between T and NK cell infiltrates and the 3-digit Miettinen score. Supplemental Figure 2. GIST TIls are mainly composed of CD4+ T cells.

Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non‐CRC) were collected for microbiome analysis. Saliva (28 non‐CRC and 94 CRC), feces (30 non‐CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM‐BC package, adjusting the P ‐values by the Holm‐Bonferroni method, revealed that Parvimonas was significantly over‐represented in feces from CRC patients ( P ‐value < 0.001) compared to healthy controls. A total of 11 Parvimonas micra isolates were obtained from the oral cavity and adenocarcinoma of CRC patients. Genome analysis identified a pair of isolates from the same patient that shared 99.2% identity, demonstrating that P. micra can translocate from the subgingival cavity to the gut. The data suggest that P. micra could migrate in a synergistic consortium with other periodontal bacteria. Metatranscriptomics confirmed that oral bacteria were more active in tumor than in non‐neoplastic tissues. We suggest that P. micra could be considered as a CRC biomarker detected in non‐invasive samples such as feces.

Abstract Background : The carcinogenesis of colorectal cancer (CRC) is a multifactorial process involving both environmental and host factors, such as human genetics or the gut microbiome, which in CRC patients appears to be enriched in oral microorganisms. The aim of this work was to investigate the presence and activity of Parvimonas micra in CRC patients. To do that, samples collected from subgingival sulcus and neoplastic lesions were used for culturomics. Then, samples from different body locations (saliva, gingival crevicular fluid, feces, non-neoplastic colon mucosa, transition colon mucosa, adenocarcinoma, adenomas, metastatic and non-neoplastic liver samples) were used for 16S rRNA metabarcoding and metatranscriptomics. Whole genome sequencing was conducted for all P. micra strains obtained. Results: Several P. micra isolates from the oral cavity and adenocarcinoma tissue from CRC patients were obtained. The comparison of oral and tumoral P. micra genomes identified that a pair of clones (PM89KC) were 99.2% identical between locations in one CRC patient, suggesting that the same clone migrated from oral cavity to the gut. The 16S rRNA metabarcoding analysis of samples from this patient revealed that P. micra cohabits with other periodontal pathogens such as Fusobacterium, Prevotella or Dialister, both in the intestine, liver and the subgingival space, which suggests that bacterial translocation from the subgingival environment to the colon or liver could be more efficient if these microorganisms travel together forming a synergistic consortium. In this way, bacteria might be able to perform tasks that are impossible for single cells. In fact, RNA-seq of the adenocarcinoma tissue confirmed the activity of these bacteria in the neoplastic tissue samples and revealed that different oral species, including P. micra, were significantly more active in the tumor compared to non-neoplastic tissue from the same individuals. Conclusion: P. micra appears to be able to translocate from the subgingival sulcus to the gut, where oral bacteria adapt to the new niche and could have a relevant role in carcinogenesis. According to our findings, periodontal disease, which increases the levels of these pathogens and facilitates their dissemination, could represent a risk factor for CRC development and P. micra could be used as a non-invasive CRC biomarker.

It is widely accepted that the presence of immune infiltrate in human tumors is considered as an attempt of the host immunosurveillance to detect and eliminate tumor cells in order to prevent tumor progression. However, the interaction of the immune system with the tumor is much more complex and highlights the controversial roles that play innate and acquired immunity in the tumor microenvironment. On the one hand, they inhibit tumor growth and dissemination, but on the other, promote cancer progression. Numerous evidences indicate that the chronic inflammatory response in the tumor microenvironment becomes a key process that polarizes immunity toward a phenotype that promotes tumor development. But, in addition, the intense immunosurveillance by the host immune system favors the selection of tumor variants with altered phenotype and reduced immunogenicity, facilitating the escape from the immune system.

The one-step nucleic acid amplification (OSNA) method allows for the quantitative evaluation of the tumor burden in resected lymph nodes (LNs) in patients with lung cancer. This technique enables to detect macro and micrometastases, facilitating the correct classification of patients for appropriate follow-up of the disease after surgery. Of 160 patients with resectable lung cancer whose LNs were examined by OSNA, H&E and CK19 IHC between July 2015 and December 2018, 110 patients with clinical stages from IA1 to IIIB were selected for follow-up. LN staging in lung cancer by pathological study led to understaging in 13.64% of the cases studied. OSNA allowed to quantify the tumor burden and establish a prognostic value. Patients with a total tumor load of ≥1650 cCP/uL were associated with a significantly increased likelihood of recurrence. Moreover, the survival of patients with <4405 cCP/uL was significantly higher than patients with ≥4405 cCP/uL. The OSNA assay is a rapid and accurate technique for quantifying the tumor burden in the LNs of lung cancer patients and OSNA quantitative data could allow to establish prognostic values for recurrence-free survival and overall survival in this type of malignancy.

Different oncogenes that act in different phases of the epithelium–adenoma–carcinoma sequence are involved in the development of colorectal carcinoma. Although this sequence is perfectly known, so far it is only possible to act on specific points of the set of known genetic alterations. Therapies aimed at neutralizing the effects of these alterations form the basis of personalized medicine or precision medicine. In this chapter, concepts closely related to these therapeutic strategies are analyzed, bringing us closer to the necessary knowledge of different types of biomarkers, key targets that currently mark treatment options, essential information to make an adequate patient selection, and the progress and challenges that arise in the short and medium term.

Cancer eradication and clinical outcome of immunotherapy depend on tumor cell immunogenicity, including HLA class I (HLA-I) and PD-L1 expression on malignant cells, and on the characteristics of the tumor microenvironment, such as tumor immune infiltration and stromal reaction. Loss of tumor HLA-I is a common mechanism of immune escape from cytotoxic T lymphocytes and is linked to cancer progression and resistance to immunotherapy with the inhibitors of PD-L1/PD-1 signaling. Here we observed that HLA-I loss in bladder tumors is associated with T cell exclusion and tumor encapsulation with stromal elements rich in FAP-positive cells. In addition, PD-L1 upregulation in HLA-I negative tumors demonstrated a correlation with high tumor grade and worse overall- and cancer-specific survival of the patients. These changes define common immuno-morphological signatures compatible with cancer immune escape and acquired resistance to therapeutic interventions across different types of malignancy. They also may contribute to the search of new targets for cancer treatment, such as FAP-expressing cancer-associated fibroblasts, in refractory bladder tumors.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p < 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and H&E. Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients.

The application to clinical practice of liquid biopsy in patients with lung cancer has led to an advance in the diagnosis and monitoring of the disease. Detection of alterations in EGFR genes related to TKI treatment in EGFR-mutated non-small cell lung cancer patients is a routine method in pathology laboratories. The primary objective of this work was to analyze the presence of EGFR mutations in cfDNA of 86 patients with lung cancer undergoing oncological treatment related to response to treatment with TKIs. Secondarily, we evaluated the dynamics of EGFR mutations, the presence of the T790M alteration and its relationship with drug resistance and analyzed by NGS molecular alterations in cfDNA of patients with discordant progression. Our results demonstrate that understanding the mutational status of patients treated with TKIs over time is essential to monitor disease progression. In this context, liquid biopsy is a fundamental key. In addition, it is not only necessary to detect EGFR mutations, but also other concomitant mutations that would be influencing the development of the disease. In this sense, we have discovered that mutations in the NF1 tumor suppressor gene could be exerting an as yet unknown function in lung cancer.

The E3 ubiquitin-ligases are important for cellular protein homeostasis and their deregulation is implicated in cancer. The E3 ubiquitin-ligase Hakai is involved in tumour progression and metastasis, through the regulation of the tumour suppressor E-cadherin. Hakai is overexpressed in colon cancer, however, the implication in colitis-associated cancer is unknown. Here, we investigated the potential role of Hakai in intestinal inflammation and cancer bowel disease. Several mouse models of colitis and associated cancer were used to analyse Hakai expression by immunohistochemistry. We also analysed Hakai expression in patients with inflamed colon biopsies from ulcerative colitis and Crohn's disease. By Hakai interactome analysis, it was identified Fatty Acid Synthase (FASN) as a novel Hakai-interacting protein. Moreover, we show that Hakai induces FASN ubiquitination and degradation via lysosome, thus regulating FASN-mediated lipid accumulation. An inverse expression of FASN and Hakai was detected in inflammatory AOM/DSS mouse model. In conclusion, Hakai regulates FASN ubiquitination and degradation, resulting in the regulation of FASN-mediated lipid accumulation, which is associated to the development of inflammatory bowel disease. The interaction between Hakai and FASN may be an important mechanism for the homeostasis of intestinal barrier function and in the pathogenesis of this disease.

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

The epithelial-mesenchymal transition (EMT) is a process with enormous relevance in tumor progression and metastasis, as well as in the acquisition of drug resistance. EMT is characterized by the loss of the epithelial phenotype and cell-substrate adhesions, and the acquisition of a motile and invasive mesenchymal phenotype. The most established marker of EMT is the loss of expression of E-cadherin, a tumor suppressor that mediates cell-cell adhesion in the epithelium. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis, and finally degradation, altering cell-cell contacts. Moreover, Hakai is important for tumor progression in different carcinomas such as colorectal cancer where Hakai is gradually increased TNM stages compared to adjacent healthy tissues. We have identified a novel small-molecule inhibitor, named Hakin-1, directed against HYB domain of Hakai domain. We show its antitumor effect in vitro, by its action on the Ecadherin ubiquitination and degradation. Furthermore, Hakin-1 reduces colony formation, proliferation, invasion and migration of colon cancer cell lines. These results suggest the potential of Hakin-1 as an effective therapy to inhibit tumor growth and carcinoma progression.

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression.

ScopePrevious work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen‐induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response.Methods and resultsAnimals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule‐associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin‐1. Also, active caspase‐3 expression, as well as poly(ADP‐ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol‐treated AIA group. Resveratrol also mitigates angiopoietin‐1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL‐1β, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression.ConclusionDietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross‐talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.