Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.
We report a case of a 68-year-old man who developed an advanced Triple-Negative Breast Cancer (TNBC) presented as a Carcinoma of Unknown Primary (CUP) at diagnoses. After progressing to first line therapy, the molecular platform EPICUP® was performed and the results suggested breast cancer as origin, although non breast primary tumor was found by imaging techniques; and the patient was treated according to this origin. During the evolution of the disease a nodule appeared in the left breast, and a biopsy confirmed the diagnosis of an infiltrating ductal carcinoma. Male breast cancer (MBC) represents an infrequent and heterogeneous disease, but TNBC in men represents less than 0.02% of total breast cancer cases. Unfortunately, there is no standard of care for MBC, and due to the development of new treatment strategies are focused on women BC, there are less opportunities to participate in clinical trials for MBC. Through the review of a clinical case, we show how the molecular platform could help in finding the origin of the CUP. Moreover, further coordinated multicenter international collaboration is necessary to conduct larger and successful therapeutic clinical trials in MBC, and specifically, male TNBC trials to better understand this rare entity and guide future management strategies.
BACKGROUND AND AIM: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. METHODS: From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. RESULTS: Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. CONCLUSIONS: Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.
In recent years, an increasing number of studies have shown that elevated expression of cyclin dependent kinase (Cdk5) contributes to the oncogenic initiation and progression of many types of cancers. In this study, we investigated the expression pattern of Cdk5 in colorectal cancer (CRC) cell lines and in a large number of tumor samples in order to evaluate its relevance in this pathogenesis and possible use as a prognostic marker. We found that Cdk5 is highly expressed and activated in CRC cell lines and that silencing of the kinase decreases their migration ability. In tumor tissues, Cdk5 is overexpressed compared to normal tissues due to a copy number gain. In patients with localized disease, we found that high Cdk5 levels correlate with poor prognosis, while in the metastatic setting, this was only the case for patients receiving an oxaliplatin-based treatment. When exploring the Cdk5 levels in the consensus molecular subtypes (CMS), we found the lowest levels in subtype 1, where high Cdk5 again was associated with a poorer prognosis. In conclusion, we confirm that Cdk5 is involved in CRC and disease progression and that it could serve as a prognostic and predictive biomarker in this disease.
Read moreOne of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.
Read moreAIMS: Pathological evaluation of colorectal carcinoma (CRC) diagnosed at stage pT1 is challenging. Nevertheless, it is crucial for treatment guiding and to determine the patient's prognosis. This study aimed to assess the interobserver variability in the histopathological evaluation of pT1 CRC. METHODS AND RESULTS: A retrospective multicentre pT1 CRC cohort study was designed (EpiT1 consortium). A task force comprising 20 experienced pathologists conducted the histopathological evaluation using digitalized haematoxylin-eosin (H&E) slides. A pilot study was performed with 10 cases, and afterwards, a consensus meeting was held to assess interobserver variability. Then, a concordance study was performed by assessing 70 new pT1 CRC cases. We used percentage agreement and Gwet's Agreement Coefficient 1 for categorical variables, and intraclass correlation coefficient (ICC) for continuous variables. In the pilot study, histological grade and perineural invasion (PNI) demonstrated 100% agreement, with good concordance for lymphovascular invasion (LVI), tumour budding (TB), poorly differentiated clusters (PDC) and margin assessment. The concordance study showed high agreement (≥90%) on histological grade, PDC, PNI and LVI. Submucosal invasion depth showed excellent reliability in the concordance study (ICC = 0.97). Notably, in both studies, the agreement of PDC was higher than for TB. Lower concordance was observed on stromal lymphocytes and the status of muscularis mucosae. CONCLUSIONS: Our results emphasize the need for standardization in evaluating pT1 CRC to improve the concordance among pathologists, and the precision of digital measurements. Moreover, the addition of PDC assessment in pT1 CRC diagnostic guidelines could help to improve the accuracy of risk stratification and reliably predict prognosis.
Read moreLymph node (LN) metastasis is an important prognostic factor in colorectal cancer (CRC). We aimed to demonstrate the presence of lymphatic vessels (LV) in the mucosa of in-situ (pTis) CRC, and of detectable tumour burden in regional LNs. This is an observational retrospective study of 39 surgically resected in situ CRCs. The number of LVs was evaluated in both pTis and normal mucosa using D2-40 immunostains. All LNs were assessed with both H&E and the One Step Nucleic Acid Amplification (OSNA) assay, and the results were correlated with clinicopathological features. D2-40 immunohistochemisty revealed LVs in the lamina propria of all pTis CRC (100%), being absent in normal mucosa. A median of 16 LNs were freshly dissected per patient, and all cases were pN0 with H&E. Molecular LN analysis with OSNA revealed the presence of low amounts of tumour burden in 11/39 (28%) cases (range 400 to 4270 CK19 mRNA copies/µL), which had no clinical consequences. This study demonstrates the presence of LVs in the lamina propria in 100% of pTis CRC, as well as the presence of low amounts of tumour burden in regional LNs, only detected by molecular methods. Given the prognostic value of LN tumour burden, its molecular quantification may help a patient's clinical management.
Read morePurpose: This work addresses the detection of Helicobacter pylori (H. pylori) in histological images with immunohistochemical staining. This analysis is a time demanding task, currently done by an expert pathologist that visually inspects the samples. Given the effort required to localise the pathogen in images, a limited number of annotations might be available in an initial setting. Our goal is to design an approach that, using a limited set of annotations, is capable of obtaining results good enough to be used as a support tool. Methods: We propose to use autoencoders to learn the latent patterns of healthy patches and formulate a specific measure of the reconstruction error of the image in HSV space. ROC analysis is used to set the optimal threshold of this measure and the percentage of positive patches in a sample that determines the presence of H. pylori. Results: Our method has been tested on an own database of 245 Whole Slide Images (WSI) having 117 cases without H. pylori and different density of the bacteria in the remaining ones. The database has 1211 annotated patches, with only 163 positive patches. This dataset of positive annotations was used to train a baseline thresholding and an SVM using the features of a pre-trained RedNet18 and ViT models. A 10-fold cross-validation shows that our method has better performance with 91% accuracy, 86% sensitivity, 96% specificity and 0.97 AUC in the diagnosis of H. pylori. Conclusion: Unlike classification approaches, our shallow autoencoder with threshold adaptation for the detection of anomalous staining is able to achieve competitive results with a limited set of annotated data. This initial approach is good enough to be used as a guide for fast annotation of infected patches.
Read moreAbstract Background Reliable biomarkers for precision medicine in metastatic colorectal cancer (mCRC) are needed. Blood biomarkers like chemokines may offer insights into overall tumor burden, yet, few prospective studies explore chemokine dynamics during treatment. This study investigates the behavior of a chemokine panel in mCRC patients during first-line oxaliplatin-based treatment, aiming to identify predictive and prognostic biomarkers. Methods Blood from oxaliplatin-treated mCRC patients was collected at three time points: before treatment (PRET), at response evaluation (EVAR), and at disease progression or last follow-up (LFUP). A custom 11-chemokine panel assessed serum chemokine levels by Luminex®, correlating them with treatment response, overall survival (OS), and progression-free survival (PFS) using the Cox proportional hazards models with the inverse probability weighting (IPW) approach. Additionally, immune system-associated gene expression was studied by Nanostring® in 15 primary tumor samples and correlated with CXCL13 expression, OS, and PFS. In silico analysis of 119 liver metastases from CRC patients, post neoadjuvant oxaliplatin-based treatment or untreated, evaluated CXCL13 expression’s correlation with immune cell infiltration, tertiary lymphoid structure (TLS) presence, OS, and PFS. Additionally, CXCL13 dynamics was studied by ELISA in 36 mCRC patients from the METIMMOX study control arm. Results Responders exhibited increased CXCL13 at EVAR, contrasting with non-responders whose levels decreased at EVAR and LFUP. Increased CXCL13 independently associated with improved PFS (median 14.5 vs. 8.9 months; HR = 0.34, p = 0.003) and OS (median 39.7 vs. 15.3 months; HR = 0.34, p = 0.003). CXCL13 expression correlated positively with an immunogenic tumor microenvironment, increased B cells, T cells (mainly CD8+) and enhanced OS. In silico , higher CXCL13 expression associated significantly with increased immune infiltration and improved OS. High CXCL13 expression was linked to the presence of TLSs, also associated with enhanced OS, especially in neoadjuvant-treated patients. Similar trends were obtained using the METIMMOX cohort. Conclusion The increase of CXCL13 levels in peripheral blood and its association with the formation of TLSs within the metastatic lesions, emerges as a potential biomarker indicative of the therapeutic efficacy in metastatic CRC patients undergoing oxaliplatin-based treatment.
Read moreMetastatic colorectal cancer (mCRC) currently lacks reliable biomarkers for precision medicine, particularly for chemotherapy-based treatments. This study examines the behavior of 11 CXC chemokines in the blood of 104 mCRC patients undergoing first-line oxaliplatin-based treatment to pinpoint predictive and prognostic markers. Serum samples were collected before treatment, at response evaluation (EVAR), and at disease progression or last follow-up. Chemokines were assessed in all samples using a Luminex® custom panel. CXCL13 levels increased at EVAR in responders, while in non-responders it decreased. Increasing levels of CXCL13 at EVAR, independently correlated with improved progression-free survival (PFS) and overall survival (OS). Nanostring® analysis in primary tumor samples showed CXCL13 gene expression's positive correlation not only with gene profiles related to an immunogenic tumor microenvironment, increased B cells and T cells (mainly CD8+) but also with extended OS. In silico analysis using RNAseq data from liver metastases treated or not with neoadjuvant oxaliplatin-based combinations, and deconvolution analysis using the MCP-counter algorithm, confirmed CXCL13 gene expression's association with increased immune infiltration, improved OS, and Tertiary Lymphoid Structures (TLSs) gene signatures, especially in neoadjuvant-treated patients. CXCL13 analysis in serum from 36 oxaliplatin-treated patients from the METIMMOX study control arm, reported similar findings. In conclusion, the increase of CXCL13 levels in peripheral blood and its association with the formation of TLSs within the metastatic lesions, emerges as a potential biomarker indicative of the therapeutic efficacy in mCRC patients undergoing oxaliplatin-based treatment • In CRC, Oxalipatin-based combinations often fail due to resistance and/or toxicity. • Lack of biomarkers complicates treatment decision-making. . • We examined serum levels of 11 CXC chemokines in patients on oxaliplatin. • Increased CXCL13 levels correlate with response, prognosis, tumor immunity and TLSs. • CXCL13 may serve as a predictive and prognostic biomarker for treatment efficacy.
Read more<p>Expression of LC-associated genes in murine mammary epithelial subpopulations</p>
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