Lozano, M.D. (María Dolores)
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- Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer(BioMed Central, 2010-06-03) Pajares, M.J. (María José); Anton, M.A. (Miguel Ángel); Pio, R. (Rubén); Lozano, M.D. (María Dolores); Gomez-Roman, J. (Javier); Agorreta, J. (Jackeline); Subirada, F. (Francesc); Durany, O. (O.); Lopez-Picazo, J.M. (José M.); Blanco, D. (Daniel); Ezponda, T. (Teresa); Montuenga-Badia, L.M. (Luis M.); Aibar, E. (Elena); Maes, T. (Tamara); Rubio, A. (Ángel)Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.
- Functional engagement of the PD-1/PD-L1 complex but not PD-l1 expression is highly predictive of patient response to immunotherapy in non-small-cell lung cancer(2023) Juaristi-Abaunz, M.A. (María Aranzazu); Gumuzio, J. (Juan); Urruticoechea, A. (Ander); Barredo-Santamaria, I. (Inmaculada); Italiano, A. (Antoine); Lozano, M.D. (María Dolores); Saiz-Camin, M. (Mónica); Claessens, N.J.M. (Niels J. M.); Geraedts, E.J. (Erica J.); Martinez-del-Prado, P. (Purificación); Martin-Algarra, S. (Salvador); Sanchez-Magraner, L. (Lissete); Elst, K. (Kim) van; Ortega, L. (Laura); Quimi, N. (Nicole); Egurrola-Izquierdo, M. (Mikel); Miles, J. (James); Calleja, V. (Veronique); Abad-Villar, M.T. (María Teresa); Segues-Merino, N. (Nerea); Gomez-Mediavilla, J. (Jenifer); Aguirre, F. (Fernando); Pikabea, F. (Fernando); Parker, C. (Charles); Applebee, C.J. (Christopher J.); Saiz-Lopez, A. (Alberto); Etxezarraga, C. (Carmen); Evans, C. (Charles); Castillo, S. (Sandra) delPURPOSE: In many cancers, the expression of immunomodulatory ligands leads to immunoevasion, as exemplified by the interaction of PD-L1 with PD-1 on tumor-infiltrating lymphocytes. Profound advances in cancer treatments have come with the advent of immunotherapies directed at blocking these immuno-suppressive ligand-receptor interactions. However, although there has been success in the use of these immune checkpoint interventions, correct patient stratification for these therapies has been challenging.MATERIALS AND METHODS: To address this issue of patient stratification, we have quantified the intercellular PD-1/PD-L1 interaction in formalin-fixed paraffin-embedded tumor samples from patients with non-small cell lung carcinoma, using a high-throughput automated quantitative imaging platform (quantitative functional proteomics [QF-Pro]).RESULTS: The multisite blinded analysis across a cohort of 188 immune checkpoint inhibitor-treated patients demonstrated the intra- and intertumoral heterogeneity of PD-1/PD-L1 immune checkpoint engagement and notably showed no correlation between the extent of PD-1/PD-L1 interaction and PD-L1 expression. Importantly, PD-L1 expression scores used clinically to stratify patients correlated poorly with overall survival; by contrast, patients showing a high PD-1/PD-L1 interaction had significantly better responses to anti-PD-1/PD-L1 treatments, as evidenced by increased overall survival. This relationship was particularly strong in the setting of first-line treatments.CONCLUSION: The functional readout of PD-1/PD-L1 interaction as a predictive biomarker for the stratification of patients with non-small-cell lung carcinoma, combined with PD-L1 expression, should significantly improve the response rates to immunotherapy. This would both capture patients excluded from checkpoint immunotherapy (high PD-1/PD-L1 interaction but low PD-L1 expression, 24% of patients) and additionally avoid treating patients who despite their high PD-L1 expression do not respond and suffer from side effects.
- An odd dancing couple. Non-small cell lung carcinoma with coexisting EGFR mutation and NTRK-1 translocation: A case report(Wiley Periodicals, 2024) Robledano, R. (Ramón); Lozano, M.D. (María Dolores)In the 21st century, there has been a dramatic shift in the diagnosis and management of non-small cell lung carcinoma (NSCLC), with an increasing use of minimally invasive tissue acquisition methods. Current treatments require morphologic subtyping and biomarker information in all cases. Determining such biomarkers is a continuously evolving field; current guidelines state that the determination of mutations on the Epidermal Growth Factor (EFGR), Kirsten Rat Sarcoma viral oncogene homolog (KRAS), Protooncogene B-Raf (BRAF), Human epidermal growth factor receptor 2 (HER2) and Anaplastic Lymphoma Kinase (ALK), genes as well as fusions on genes such as ROS ProtoOncogene 1, Receptor Tyrosine Kinase (ROS1), MET proto oncogene, receptor tyrosine kinase (MET), RET proto-oncogene (RET), and the Neurotrophic Tyrosine Receptor Kinase (NTRK) family is mandatory. While analyzing such alterations, some of them were first reported to be mutually exclusive, although in recent years, it has been shown otherwise in some of these cases. Moreover, so was the case with the concomitant expression of NTRK fusions and EGFR mutations. We present a case report of a patient with concomitant EGFR mutation and NTRK1 fusion.
- TMPRSS4: a novel tumor prognostic indicator for the stratification of stage IA tumors and a liquid biopsy biomarker for NSCLC patients(MDPI AG, 2019) Behrens, C. (C.); Pajares, M.J. (María José); Expósito, F. (Francisco); Pio, R. (Rubén); Sainz, C. (Cristina); Lozano, M.D. (María Dolores); Remirez, A. (Ana); Jantus-Lewintre, E. (Eloisa); Camps, C. (Carlos); Montuenga-Badia, L.M. (Luis M.); Villalba-Esparza, M. (María); Redrado, M. (Miriam); Andrea, C.E. (Carlos Eduardo) de; Wistuba, I.I. (Ignacio I.); Calvo-González, A. (Alfonso)Relapse rates in surgically resected non-small-cell lung cancer (NSCLC) patients are between 30% and 45% within five years of diagnosis, which shows the clinical need to identify those patients at high risk of recurrence. The eighth TNM staging system recently refined the classification of NSCLC patients and their associated prognosis, but molecular biomarkers could improve the heterogeneous outcomes found within each stage. Here, using two independent cohorts (MDA and CIMA-CUN) and the eighth TNM classification, we show that TMPRSS4 protein expression is an independent prognostic factor in NSCLC, particularly for patients at stage I: relapse-free survival (RFS) HR, 2.42 (95% CI, 1.47–3.99), p < 0.001; overall survival (OS) HR, 1.99 (95% CI, 1.25–3.16), p = 0.004). In stage IA, high levels of this protein remained associated with worse prognosis (p = 0.002 for RFS and p = 0.001 for OS). As TMPRSS4 expression is epigenetically regulated, methylation status could be used in circulating tumor DNA from liquid biopsies to monitor patients. We developed a digital droplet PCR (ddPCR) method to quantify absolute copy numbers of methylated and unmethylated CpGs within the TMPRSS4 and SHOX2 (as control) promoters in plasma and bronchoalveolar lavage (BAL) samples. In case-control studies, we demonstrated that TMPRSS4 hypomethylation can be used as a diagnostic tool in early stages, with an AUROC of 0.72 (p = 0.008; 91% specificity and 52% sensitivity) for BAL and 0.73 (p = 0.015; 65% specificity and 90% sensitivity) for plasma, in early stages. In conclusion, TMPRSS4 protein expression can be used to stratify patients at high risk of relapse/death in very early stages NSCLC patients. Moreover, analysis of TMPRSS4 methylation status by ddPCR in blood and BAL is feasible and could serve as a non-invasive biomarker to monitor surgically resected patients.
- Identification of importin (IPO-8) as the most accurate reference gene for the clinicopathological analysis of lung specimens(BioMed Central, 2008-11-17) Pajares, M.J. (María José); Pio, R. (Rubén); Lozano, M.D. (María Dolores); Gomez-Roman, J. (Javier); Agorreta, J. (Jackeline); Rodriguez, M.J. (María José); Sanchez, B.A. (Blas A.); Blanco, D. (Daniel); Montuenga-Badia, L.M. (Luis M.); Valles, I. (Iñaki); Nguewa, P.A. (Paul Alain); Calvo-González, A. (Alfonso)Abstract Background: The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results: We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RTPCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion: Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.
- Elevated levels of the complement activation product c4d in bronchial fluids for the diagnosis of lung cancer(Public Library of Science, 2015) Pajares, M.J. (María José); Garcia, J. (Javier); Pio, R. (Rubén); Lozano, M.D. (María Dolores); Schmidt, B. (Bernd); Fleischhacker, M. (M.); Nadal, E. (Ernest); Montuenga-Badia, L.M. (Luis M.); Razquin, C. (Cristina); Paz-Ares, L. (Luis); Ajona, D. (Daniel); Pastor, M.D. (María Dolores); Cardenal, F. (F.); Zulueta, J. (Javier)Molecular markers in bronchial fluids may contribute to the diagnosis of lung cancer. We previously observed a significant increase of C4d-containing complement degradation fragments in bronchoalveolar lavage (BAL) supernatants from lung cancer patients in a cohort of 50 cases and 22 controls (CUN cohort). The present study was designed to determine the diagnostic performance of these complement fragments (hereinafter jointly referred as C4d) in bronchial fluids. C4d levels were determined in BAL supernatants from two independent cohorts: the CU cohort (25 cases and 26 controls) and the HUVR cohort (60 cases and 98 controls). A series of spontaneous sputum samples from 68 patients with lung cancer and 10 controls was also used (LCCCIO cohort). Total protein content, complement C4, complement C5a, and CYFRA 21-1 were also measured in all cohorts. C4d levels were significantly increased in BAL samples from lung cancer patients. The area under the ROC curve was 0.82 (95%CI = 0.71-0.94) and 0.67 (95%CI = 0.58-0.76) for the CU and HUVR cohorts, respectively. In addition, unlike the other markers, C4d levels in BAL samples were highly consistent across the CUN, CU and HUVR cohorts. Interestingly, C4d test markedly increased the sensitivity of bronchoscopy in the two cohorts in which cytological data were available (CUN and HUVR cohorts). Finally, in the LCCCIO cohort, C4d levels were higher in sputum supernatants from patients with lung cancer (area under the ROC curve: 0.7; 95%CI = 0.56-0.83). In conclusion, C4d is consistently elevated in bronchial fluids from lung cancer patients and may be used to improve the diagnosis of the disease.
- EUELC project: a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration. European Respiratory(European Respiratory Society, 2009-05-15) Pajares, M.J. (María José); Niklinski, J. (J.); Vignaud, J.M. (J.M.); Lozano, M.D. (María Dolores); EUELC; Niaz, A. (A.); Hainaut, P. (P.); Roz, L. (Luca); Vesin, A. (Aurelien); Liloglou, T. (Triantafillos); Elborn, J. (J.S.); Field, J.K. (J. K.); Prinsen, C. (C.); Gosney, J.R. (J. R.); Giles, T. (T.); Montuenga-Badia, L.M. (Luis M.); Brambilla, E. (E.); Sozzi, G. (Gabriella); Magee, N.D. (N.D.); Bryan, J. (J.); Cassidy, A. (A.); Thunnissen, F.B. (Frederick B.); Martinet, Y. (Y.); Risch, A. (A.); O'Byrne, K.J. (K.J.); Snijders, P.J. (P.J.); Harrison, D.J. (D. J.); Timsit, J.F. (J.F.); Becker, H. (H.D.); Smit, E.F. (E.F.); Brambilla, C. (C.)The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular–pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARb genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes.
- Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study(Elsevier BV, 2019) Gonzalez-Larriba, J.L. (José Luis); Plaza, M.L. (María Luz); Rodríguez-Abreu, D. (Delvys); Domine, M. (Manuel); Lozano, M.D. (María Dolores); García-González, J (Jorge); Enguita, A.B. (Ana Belén); Gomez-Roman, J. (Javier); Lázaro-Quintela, M. (Martín); Aguiar, D. (David); Saiz, M. (Mónica); Felip, E. (Enriqueta); Muriel, A. (Alfonso); López-Brea, M. (Marta); Mancheño, N. (Nuria); Arriola, E. (Edurne); Conde, E. (Esther); Garrido, P. (Pilar); Atienza-Cuevas, L. (Lidia); González-Piñeiro, A. (Ana); Jiménez, B. (Beatriz); Gurpide, A. (Alfonso); Martínez-Turrillas, R. (Rebeca); Arriola-Arellano, E. (Esperanza); Esteban-Rodríguez, I. (Isabel); Teixidó, C. (Cristina); Company, A. (Amparo); Aranda, I. (Ignacio); Hernández, S. (Susana); Moran, T. (Teresa); Castro, J. (Javier) de; Camacho, C. (Carmen); Benito, A. (Amparo); Álvarez, R. (Ramiro); Paz-Ares, L. (Luis); Mate, J.L. (Jose Luis); Sansano, I. (Irene); Reguart, N. (Noemi); López-Ríos, F. (Fernando); Artal, Á. (Ángel); Angulo, B. (Bárbara); Juan, O. (Oscar); García, F. (Felip); Abdulkader, I. (Ihab); Salido, M. (Marta); Sanz, J. (Julián); Collazo-Lorduy, A. (Ana); Rojo-Todo, F. (Federico); Insa, A. (Amelia); Pijuan, L. (Lara); Massuti, B. (Bartomeu); Azcona, E. (Eider)Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.
- Modification of breast cancer milieu with chemotherapy plus dendritic cell vaccine: an approach to select best therapeutic strategies(2023) Hato-Álvaro, L. (Laura); Espinos, J. (Jaime); Lozano, M.D. (María Dolores); Pérez-Solans, B. (Belén); Lopez-Diaz-de-Cerio, A. (Ascensión); Córdoba-Iturriagagoitia, A. (Alicia); Santisteban, M. (Marta); Inoges, S. (Susana); Guillen-Grima, F. (Francisco); Mejías-Sosa, L.D. (Luis D.); Idoate, M.A. (Miguel Ángel); Sala-Elarre, P. (Pablo); Cruz, S. (S.) de la; López-Janeiro, Á. (Álvaro)Background: The addition of dendritic cell vaccines (DCV) to NAC could induce immune responses in those patients with residual disease (RD) by transforming the tumor microenvironment. Methods: Core diagnostic biopsies and surgical specimens from 80 patients (38 in the vaccinated group plus NAC (VG) and 42 in the control group (CG, treated only with NAC) were selected. We quantify TILs (CD8, CD4 and CD45RO) using immunohistochemistry and the automated cellular imaging system (ACIS III) in paired samples. Results: A CD8 rise in TNBC samples was observed after NAC plus DCV, changing from 4.48% in the biopsy to 6.70% in the surgical specimen, not reaching statistically significant differences (p = 0.11). This enrichment was seen in up to 67% of TNBC patients in the experimental arm as compared with the CG (20%). An association between CD8 TILs before NAC (4% cut-off point) and pathological complete response in the VG was found in the univariate and multivariate analysis (OR = 1.41, IC95% 1.05-1.90; p = 0.02, and OR = 2.0, IC95% 1.05-3.9; p = 0.03, respectively). Conclusion: Our findings suggest that patients with TNBC could benefit from the stimulation of the antitumor immune system by using DCV together with NAC.
- Assessment of indeterminate pulmonary nodules detected in lung cancer screening: Diagnostic accuracy of FDG PET/CT(Elsevier BV, 2016) Torre, W. (Wenceslao); Campo, A. (Arantza); Lozano, M.D. (María Dolores); Bastarrika, G. (Gorka); Sancho, L. (Lidia); Richter, J.A. (José Ángel); Sanchez-Salcedo, P. (Pablo); Torres, J.P. (Juan P.) de; Garcia-Velloso, M. J. (María José); Alcaide, A.B. (Ana Belén); Nuñez-Cordoba, J.M. (Jorge M.); Zulueta, J. (Javier)Background: A major drawback of lung cancer screening programs is the high frequency of false-positive findings on computed tomography (CT). We investigated the accuracy of selective 2-[fluorine-18]- fluoro-2-deoxy-d-glucose (FDG) Positron Emission Tomography/Computed Tomography (PET/CT) scan in assessing radiologically indeterminate lung nodules detected in lung cancer screening. Methods: FDG PET/CT was performed to characterize 64 baseline lung nodules >10 mm and 36 incidence nodules detected on low-dose CT screening in asymptomatic current or former smokers (83 men, age range 40–83 years) at high risk for lung cancer. CT images were acquired without intravenous contrast. Nodules were analyzed by size, density, and metabolic activity and visual scored on a 5-point scale for FDG uptake. Nodules were classified as negative for malignancy when no FDG uptake was observed, or positive when focal uptake was observed in the visual analysis, and the maximum standardized uptake value (SUVmax) was measured. Final diagnosis was based on histopathological evaluation or at least 24 months of follow-up. Results: A total of 100 nodules were included. The prevalence of lung cancer was 1%. The sensitivity, specificity, NPV and PPV of visual analysis to detect malignancy were 84%, 95%, 91%, and 91%, respectively, with an accuracy of 91% (AUC 0.893). FDG PET/CT accurately detected 31 malignant tumors (diameters 9–42 mm, SUVmax range 0.6–14.2) and was falsely negative in 6 patients. With SUVmax thresholds for malignancy of 1.5, 2, and 2.5, specificity was 97% but sensitivity decreased to 65%, 49%, and 46% respectively, and accuracy decreased to 85%, 79%, and 78% respectively (AUC 0.872). Conclusions: The visual analysis of FDG PET/CT scan is highly accurate in characterizing indeterminate pulmonary nodules detected in lung cancer screening with low-dose CT. Semi-quantitative analysis does not improve the accuracy of FDG PET/CT over that obtained with a qualitative method for lung nodule characterization.