Mecanismos de resistencia primaria y adquirida a trastuzumab en cáncer de mama HER2 positivo

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 05-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 05-01-201

Thymidylate synthase expression as a predictive biomarker of pemetrexed sensitivity in advanced non-small cell lung cancer

Background: Although it has been suggested that a high level of thymidylate synthase (TYMS) gene expression in malignant tumors is related to reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with the drug. The purpose of this study was to quantitatively assess the impact of TYMS gene expression in tumor cells as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer (NSCLC) treated at our institution. Methods: Sixty-two NSCLC patients were included in this study: 16 patients received platins-pemetrexed as first-line NSCLC, and 46 pemetrexed in monotherapy as second- or subsequent-line treatment. Total mRNA was isolated and the expression of TYMS was analyzed by RT-qPCR. TYMS levels were calibrated against expression in normal lung tissue. Results: TYMS overexpression was detected in 61 % of patients and low expression in 39 %. The response rate for patients with low TYMS expression was 0.29 compared with 0.03 in patients with overexpression (P = 0.025). A significant benefit was observed in patients with low expression both in time to progression (average TTP = 56 vs. 23 months, P = 0.001) and in overall survival (average OS = 60 vs. 25 months, P = 0.002). Conclusions: TYMS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced NSCLC patientsThe present work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO) (AES Program, grant PI12/01552); the Ministerio de Sanidad (Cancer Network); and the Comunidad de Madrid (S2010/BMD-2344). The Fundacion Jimenez Diaz Biobank is funded by a grant from the MINECO (Instituto de Salud Carlos III, RETICS Red de Biobancos, with FEDER funds, RD09/0076/00101). S.Z. and C.C. are supported by grants from the same Biobanks initiativ

Activation of MET pathway predicts poor outcome to cetuximab in patients with recurrent or metastatic head and neck cancer

Background: Activation of the MET oncogene promotes tumor growth, invasion and metastasis in several tumor types. Additionally, MET is activated as a compensatory pathway in the presence of EGFR blockade, thus resulting in a mechanism of resistance to EGFR inhibitors. Methods: We have investigated the impact of HGF and MET expression, MET activation (phosphorylation), MET gene status, and MET-activating mutations on cetuximab sensitivity in recurrent or metastatic squamous cell carcinoma of the head and neck (HNSCC) patients. Results: A single-institution retrospective analysis was performed in 57 patients. MET overexpression was detected in 58 % patients, MET amplification in 39 % and MET activation (p-MET) in 30 %. Amplification was associated with MET overexpression. Log-rank testing showed significantly worse outcomes in recurrent/metastatic, MET overexpressing patients for progression-free survival and overall survival. Activation of MET was correlated with worse PFS and OS. In multivariate logistic regression analysis, p-MET was an independent prognostic factor for PFS. HGF overexpression was observed in 58 % patients and was associated with MET phosphorylation, suggesting a paracrine activation of the receptor. Conclusions: HGF/MET pathway activation correlated with worse outcome in recurrent/metastatic HNSCC patients. When treated with a cetuximab-based regimen, these patients correlated with worse outcome. This supports a dual blocking strategy of HGF/MET and EGFR pathways for the treatment of patients with recurrent/metastatic HNSCCThe present work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO) (AES Program, grant PI12/01552); the Ministerio de Sanidad (Cancer Network); the Comunidad de Madrid (S2010/BMD-2344). The Fundacion Jimenez Diaz Biobank is funded by a grant from the MINECO (Instituto de Salud Carlos III, RETICS Red de Biobancos, with FEDER funds, RD09/0076/00101). S.Z. and C.C. are supported by grants from the same Biobanks initiativ

Basement membrane-rich Organoids with functional human blood vessels are permissive niches for human breast cancer metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention

Low microRNA-19b expression shows a promising clinical impact in locally advanced rectal cancer

The standard treatment for patients with locally advanced colorectal cancer (LARC) is neoadjuvant 5-fluorouracil (5-FU) based chemoradiotherapy (CRT) followed by surgical mesorec-tal excision. However, the lack of response to this preoperative treatment strongly compromises patient outcomes and leads to surgical delays and undesired toxicities in those non-responder cases. Thus, the identification of effective and robust biomarkers to predict response to preoperative CRT represents an urgent need in the current clinical management of LARC. The oncomiR microRNA-19b (miR-19b) has been reported to functionally play oncogenic roles in colorectal cancer (CRC) cells as well as regulate 5-FU sensitivity and determine outcome in CRC patients. However, its clinical impact in LARC has not been previously investigated. Here, we show that miR-19b deregulation is a common event in this disease, and its decreased expression significantly associates with lower tumor size after CRT (p = 0.003), early pathological stage (p = 0.003), and absence of recurrence (p = 0.001) in LARC patients. Interestingly, low miR-19b expression shows a predictive value of better response to neoajuvant CRT (p < 0.001), and the subgroup of LARC patients with low miR-19b levels have a markedly longer overall (p = 0.003) and event-free survival (p = 0.023). Finally, multivariate analyses determined that miR-19b independently predicts both patient outcome and response to preoperative CRT, highlighting its potential clinical usefulness in the management of LARC patientsThis research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER”. M.S-A. is supported by “Fundación Cristina Rábago de Jiménez Díaz

Validation of microrna‐199b as a promising predictor of outcome and response to neoadjuvant treatment in locally advanced rectal cancer patients

The absence of established predictive markers with value to anticipate response to neoadjuvant 5‐fluorouracil (5‐FU)‐based chemoradiotherapy (CRT) represents a current major challenge in locally advanced rectal cancer (LARC). The tumor suppressor microRNA (miR)‐199b has been reported to play a key role determining 5‐FU sensitivity of colorectal cancer cells through the regulation of several signaling pathways, and has emerged as a novel molecular target to overcome the 5‐FU resistant phenotype. Moreover, miR‐199b downregulation was described as a common alteration that predicts lack of response to preoperative CRT in LARC but this issue needs to be confirmed in independent larger cohorts. Here, we evaluate the clinical impact of miR‐199b in LARC and perform additional analyses to further clarify its potential relevance as novel marker in this disease. Thus, miR‐199b expression was quantified by real‐time‐PCR in a cohort of 185 LARC patients, observing this miR downregulated in 22.2% of cases and significantly associated with higher tumor size (p= 0.026) and positive lymph node after CRT (p= 0.005), and higher pathological stage (p= 0.004). Notably, this alteration showed a strong independent predictive value of poor pathological response to neoadjuvant CRT (p= 0.004). Moreover, the subgroup of cases with low miR‐199b levels had a markedly shorter overall (p< 001) and event‐free survival (p< 0.001), and multivariate analyses showed that miR‐199b deregulation represents an independent prognosticator for patient outcome in LARC. Interestingly, the prognostic impact of this miR was strongly significant in both younger and elderly patients, and was very effective determining patient recurrence (p= 0.004). Finally, we compared miR‐199b expression profiles in a set of cases with pre and post‐treatment samples available, observing that only a minimal response leads to miR‐199b increase levels, further suggesting its potential clinical and therapeutic relevance as a promising marker and novel molecular target for the management of LARC.This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER”. M.S-A. is supported by a predoctoral research grant funded by “Fundación Conchita Rábago de Jiménez Díaz”

Microrna-199b downregulation confers resistance to 5-fluorouracil treatment and predicts poor outcome and response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients

Neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy followed by mesorectal excision is the current standard treatment in locally advanced rectal cancer (LARC) and the lack of complete response represents a major problem that compromises long-term patient survival. However, there is a lack of robust established markers predictive of response to this preoperative treatment available in the clinical routine. The tumor suppressor microRNA (miR)-199b directly targets the PP2A inhibitor SET, which has been involved in 5-FU resistance, and its downregulation has been found to correlate with poor outcome in metastatic colorectal cancer. Here, we studied the functional effects of miR-199b on 5-FU sensitivity after its ectopic modulation, and its expression was quantified by real-time-PCR in a cohort of 110 LARC patients to evaluate its potential clinical significance. Interestingly, our findings demonstrate that miR-199b enhances the sensitivity of colorectal cancer cells to 5-FU in a SET-dependent manner, and that both miR-199b overexpression and SET inhibition are able to overcome resistance to this drug using an acquired 5-FU-resistant model. MiR-199b was found downregulated in 26.4% of cases and was associated with positive lymph node levels after chemoradiotherapy (CRT, p = 0.007) and high pathological stage (p = 0.029). Moreover, miR-199b downregulation determined shorter overall (p = 0.003) and event-free survival (p = 0.005), and was an independent predictor of poor response to preoperative CRT (p = 0.004). In conclusion, our findings highlight the clinical impact of miR-199b downregulation predicting poor outcome and pathological response in LARC, and suggest the miR-199b/SET signaling axis as a novel molecular target to prevent the development of resistance to 5-FU treatment.This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.This work was funded by the Ministerio de Ciencia, Innovacion y Universidades of Spain and Instituto de Salud Carlos III (PI16/00528, PI16/00946, PI16/01207 and PI16/01276), co-funded by the Spanish Biobank Network (PT13/0010/0030, PT17/0015/0001, PT17/0015/0021, PT17/0015/0049, PT17/0015/0018, PT17/0015/0002, PT17/0015/0016, PT17/0015/0038, PT17/0015/0027, PT17/0015/0004, PT17/0015/0047, PT17/0015/0014, PT17/0015/0041, and PT17/0015/0006), European Regional Development Fund (FEDER) "A way to make Europe" and granted by Conselleria d'Innovacio, Recerca i Turisme del Govern de les Illes Balears (TEC/002/2017).S

Aplikasi Herbisida 2,4-d Dan Penoxsulam Pada Pertumbuhan Dan Hasil Tanaman Padi Sawah (Oryza Sativa L.)

Salah satu teknik budidaya untuk meningkatkan produksi tanaman padi sawah yaitu dengan mengurangi persaingan antara tanaman dengan gulma. Pengendalian dengan kimiawi merupakan salah satu cara mengurangi pertumbuhan gulma di pertanaman padi. Cara kimiawi merupakan cara yang praktis, efektif dan efisien untuk mengendalikan gulma. Penelitian ini bertujuan untuk mempelajari pengaruh dari aplikasi herbisida 2,4-D dan penoxsulam dalam meningkatkan pertumbuhan dan hasil padi sawah serta menentukan dosis aplikasi herbisida 2,4-D dan penoxsulam baik secara tunggal maupun campuran dalam mengendalikan gulma pada tanaman padi sawah. Penelitian telah dilaksanakan pada bulan Maret-Juli 2014 di Desa Campurasri, Ngawi. Penelitian menggunakan Rancangan Acak Kelompok sederhana, dengan menempatkan 11 perlakuan yaitu H1 : kontrol herbisida 2,4-D; H2 : 2,4-D 11,25 kg ha-1; H3 : 2,4-D 22,5 kg ha-1; H4 : 2,4-D 33,75 kg ha-1; H5 : kontrol herbisida penoxsulam; H6 : penoxsulam 200 ml ha-1; H7 : penoxsulam 400 ml ha-1; H8 : penoxsulam 600 ml ha-1; H9 : 2,4-D 11,25 kg ha-1 dan penoxsulam 200 ml ha-1; H10 : 2,4-D 22,5 kg ha-1 dan penoxsulam 400 ml ha-1; H11 : 2,4-D 33,75 kg ha-1 dan penoxsulam 600 ml ha-1. Hasil penelitian menunjukkan bahwa perlakuan herbisida 2,4-D 11,25 kg ha-1 dan penoxsulam 200 ml menghasilkan bobot kering total tanaman dengan peningkatan sebesar 34,62 % dibandingkan dengan kontrol. Pada produksi tanaman padi peningkatan terjadi sebesar 29,77 % pada perlakuan herbisida 2,4-D 33,75 kg ha-1 dan penoxsulam 600 ml dibandingkan dengan kontrol

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer