Nuestros investigadores

Carlos Eduardo De Andrea 

Publicaciones científicas más recientes (desde 2010)

Autores: Lozano Escario, María Dolores (Autor de correspondencia); Landa, A. ; García Tobar, Laura; et al.
ISSN 8755-1039  Vol. 48  Nº 9  2020  págs. 827 - 832
Desmoplastic small round cell tumor (DSRCT) is rare and a highly aggressive neoplasm that typically involves the soft tissues of the abdomen or pelvis in children or young adults, showing a male predilection. Although it can occurs over a wide age range, the peak incidence is in the third decade of life. DSRCT usually shows widespread abdominal serosal involvement, and overall patient survival is poor. On the other hand, extra-abdominal DSRCT is very rare. DSRCT in major salivary glands has been reported, but it is extremely rare. In the majority of reported series diagnosis is made by the histological analysis of FFPE tissues together with immunohistochemistry (IHC) and molecular analysis, particularly the demonstration of chromosomal translocation involving EWSR1. Very few cases have been diagnosed so far by Fine Needle Aspiration (FNA) cytology. Moreover ancillary studies have been performed in all reported cases in FFPE samples. There is still controversy and lack of consensus regarding the suitability of cytological samples especially smears for immunocytochemical (ICC) and fluorescence in situ hybridization (FISH), what makes its standardization difficult. We report a case of a primary DSRCT of parotid gland in a 17-year-old male diagnosed by FNA cytology. The cytomorphological diagnosis was coupled with ICC and FISH analysis performed on stained smears. We emphasize the feasibility and reliability of cytological smears for the application of immunocytochemical and molecular techniques.
Autores: De Andrea, Carlos Eduardo; Pérez Gracia, José Luis; Castañón Álvarez, Eduardo; et al.
ISSN 2162-402X  Vol. 9  Nº 1  2020  págs. 1760676
Checkpoint inhibitors have improved the survival of patients with advanced tumors and show a manageable toxicity profile. However, auto-immune colitis remains a relevant side effect, and combinations of anti-PD1/PDL1 and anti-CTLA-4 increase its incidence and severity. Here, we report the case of a 50-year-old patient diagnosed with stage IV cervical cancer that relapsed following radical surgery, external radiation/brachytherapy and standard chemotherapy. She was subsequently treated with Nivolumab and Ipilimumab combination and developed grade 2 colitis presenting a dissociation between endoscopic and pathological findings. At cycle 10 the patient reported grade 3 diarrhea and abdominal discomfort, without blood or mucus in the stools. Immunotherapy was withheld and a colonoscopy was performed, showing normal mucosa in the entire colon. Puzzlingly, histologic evaluation of randomly sampled mucosal biopsy of the distal colon showed an intense intraepithelial lymphocyte infiltration with crypt loss and some regenerating crypts with a few apoptotic bodies set in a chronically inflamed lamina propria, consistent with the microscopic diagnosis of colitis. Treatment with methylprednisolone 2 mg/¿kg was initiated which led to a decrease in the number of stools to grade 1. Additional investigations to exclude other causes of diarrhea rendered negative results. The patient experienced a major partial response and, following the resolution of diarrhea, she was re-challenged again with immunotherapy, with the reappearance of grade 2 diarrhea, leading to permanent immunotherapy interruption. We conclude and propose that performing random colonic biopsies should be considered in cases of immune checkpoint-associated unexplained diarrhea, even when colonoscopy shows macroscopically normal colonic mucosa inflammatory lesions.
Autores: Otano Andrés, Itziar (Autor de correspondencia); Alvarez Rodriguez, Maite; Minute, L.; et al.
ISSN 1838-7640  Vol. 10  Nº 10  2020  págs. 4481 - 4489
Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.
Autores: Teijeira Sánchez, Álvaro (Autor de correspondencia); Garasa, S.; Gato Cañas, María; et al.
ISSN 1074-7613  Vol. 52  Nº 5  2020  págs. 856 - 871.E8
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
Autores: Failmezger, H.; Muralidhar, S.; Rullan, A. ; et al.
ISSN 0008-5472  Vol. 80  Nº 5  2020  págs. 1199 - 1209
Despite the advent of immunotherapy, metastatic melanoma represents an aggressive tumor type with a poor survival outcome. The successful application of immunotherapy requires in-depth understanding of the biological basis and immunosuppressive mechanisms within the tumor microenvironment. In this study, we conducted spatially explicit analyses of the stromal-immune interface across 400 melanoma hematoxylin and eosin (H&E) specimens from The Cancer Genome Atlas. A computational pathology pipeline (CRImage) was used to classify cells in the H&E specimen into stromal, immune, or cancer cells. The estimated proportions of these cell types were validated by independent measures of tumor purity, pathologists' estimate of lymphocyte density, imputed immune cell subtypes, and pathway analyses. Spatial interactions between these cell types were computed using a graph-based algorithm (topological tumor graphs, TTG). This approach identified two stromal features, namely stromal clustering and stromal barrier, which represented the melanoma stromal microenvironment. Tumors with increased stromal clustering and barrier were associated with reduced intratumoral lymphocyte distribution and poor overall survival independent of existing prognostic factors. To explore the genomic basis of these TTG-derived stromal phenotypes, we used a deep learning approach integrating genomic (copy number) and transcriptomic data, thereby inferring a compressed representation of copy number-driven alterations in gene expression. This integrative analysis revealed that tumors with high stromal clustering and barrier had reduced expression of pathways involved in naive CD4 signaling, MAPK, and PI3K signaling. Taken together, our findings support the immunosuppressive role of stromal cells and T-cell exclusion within the vicinity of melanoma cells. Significance: Computational histology-based stromal phenotypes within the tumor microenvironment are significantly associated with prognosis and immune exclusion in melanoma.
Autores: Ajona Martínez-Polo, Daniel; Ortiz Espinosa, Sergio; Lozano Moreda, Teresa; et al.
ISSN 2662-1347  Vol. 1  2020  págs. 75 - 85
Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1¿programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.
Autores: Recalde Zamacona, Borja; García Tobar, Laura; Argueta Morales, Allan; et al.
Revista: THORAX
ISSN 1468-3296  Vol. 75  Nº 12  2020  págs. 1116 - 1118
In December 2019, an outbreak of severe acute respiratory syndrome associated to SARS-CoV2 was reported in Wuhan, China. To date, little is known on histopathological findings in patients infected with the new SARS-CoV2. Lung histopathology shows features of acute and organising diffuse alveolar damage. Subtle cellular inflammatory infiltrate has been found in line with the cytokine storm theory. Medium-size vessel thrombi were frequent, but capillary thrombi were not present. Despite the elevation of biochemical markers of cardiac injury, little histopathological damage could be confirmed. Viral RNA from paraffin sections was detected at least in one organ in 90% patients.
Autores: Baraibar Argota, Iosune; Román Moreno, Marta; Rodríguez Remírez, María; et al.
Revista: CANCERS
ISSN 2072-6694  Vol. 12  Nº 11  2020  págs. 31-69
Autores: Lozano Escario, María Dolores; García Tobar, Laura; Abengozar Muela, Marta; et al.
ISSN 0023-6837  Vol. 100  Nº Supl. 1  2020  págs. 392
Autores: Lozano Escario, María Dolores; Abengozar Muela, Marta; Alvarez, M.; et al.
ISSN 0023-6837  Vol. 100  Nº Supl. 1  2020  págs. 391 - 392
Autores: Lozano Escario, María Dolores; Abengozar Muela, Marta; Alvarez, M.; et al.
ISSN 0023-6837  Vol. 100  Nº Supl. 1  2020  págs. 390 - 391
Autores: Pisapia, P.; Malapelle, U.; Roma, G.; et al.
ISSN 1934-662X  Vol. 127  Nº 5  2019  págs. 285 - 296
Background Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 x 10(5)). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.
Autores: Lozano Escario, María Dolores; Abengozar Muela, Marta; Echeveste, José Ignacio; et al.
ISSN 1934-662X  Vol. 127  Nº 7  2019  págs. 470 - 480
Background Programmed death-ligand 1 (PD-L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. The current study evaluated the feasibility and efficacy of PD-L1 immunostaining and quantitation on direct Papanicolaou-stained cytological smears compared with formalin-fixed paraffin-embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona). Methods PD-L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC). Results In 57 sets of samples, both PD-L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin-fixed paraffin-embedded samples was observed in 97.3% of the cases. Conclusions The quantification of PD-L1 expression on direct Papanicolaou-stained cytology smears is feasible and reliable for both PD-L1 assays.
Autores:  Perez-Ruiz, E.; Cordeiro Minute, Luna Ridan; Otano Andrés, Itziar; et al.
Revista: NATURE
ISSN 0028-0836  Vol. 569  Nº 7756  2019  págs. 428 - 432
Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
Autores: Villalba Esparza, María; Expósito Rincón, Francisco; Pajares Villandiego, María José; et al.
ISSN 2077-0383  Vol. 8  Nº 12  2019  págs. E2134
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.
Autores: Villalba Esparza, María; Redín Cabodevilla, María Esther; Expósito Rincón, Francisco; et al.
ISSN 2045-2322  Vol. 9  2019  págs. 15400
Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (similar to 40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4.
Autores: Martínez Valbuena, Iván; Valentí Azcárate, Rafael; Amat-Villegas, I.; et al.
ISSN 0364-5134  Vol. 86  Nº 4  2019  págs. 539 - 551
Objective Alzheimer disease (AD) is the leading cause of dementia, and although its etiology remains unclear, it seems that type 2 diabetes mellitus (T2DM) and other prediabetic states of insulin resistance could contribute to the appearance of sporadic AD. As such, we have assessed whether tau and beta-amyloid (A beta) deposits might be present in pancreatic tissue of subjects with AD, and whether amylin, an amyloidogenic protein deposited in the pancreas of T2DM patients, might accumulate in the brain of AD patients. Methods We studied pancreatic and brain tissue from 48 individuals with no neuropathological alterations and from 87 subjects diagnosed with AD. We examined A beta and tau accumulation in the pancreas as well as that of amylin in the brain. Moreover, we performed proximity ligation assays to ascertain whether tau and/or A beta interact with amylin in either the pancreas or brain of these subjects. Results Cytoplasmic tau and A beta protein deposits were detected in pancreatic beta cells of subjects with AD as well as in subjects with a normal neuropathological examination but with a history of T2DM and in a small cohort of control subjects without T2DM. Furthermore, we found amylin deposits in the brain of these subjects, providing histological evidence that amylin can interact with A beta and tau in both the pancreas and hippocampus. Interpretation The presence of both tau and A beta inclusions in pancreatic beta cells, and of amylin deposits in the brain, provides new evidence of a potential overlap in the mechanisms underlying the pathogenesis of T2DM and AD. ANN NEUROL 2019
Autores: Schalper, K. A. ; Rodriguez-Ruiz, M. E.; Diez Valle, Ricardo; et al.
ISSN 1078-8956  Vol. 25  Nº 3  2019  págs. 470 - 476
Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival1,2. Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3¿cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.
Autores: Expósito Rincón, Francisco; Villalba. M.; Redrado Jordán, Miriam; et al.
ISSN 0304-3835  Vol. 453  2019  págs. 21 - 33
High mortality rates caused by NSCLC show the need for the identification of novel therapeutic targets. In this study we have investigated the biological effects and molecular mechanisms elicited by TMPRSS4 in NSCLC. Overexpression of TMPRSS4 in LKR13¿cells increased malignancy, subcutaneous tumor growth and multiorganic metastasis. In conditional knock-down (KD) experiments, abrogation of TMPRSS4 in H358 and H2170¿cells altered proliferation, clonogenicity, tumor engraftment and tumor growth. Reduction in S and G2/M phases of the cell cycle, decreased BrdU incorporation and increased apoptosis was also found. Transcriptomic analysis in KD cells revealed downregulation of genes involved in DNA replication, such as MCM6, TYMS and CDKN1A (p21). In patients, expression of a signature of MCM6/TYMS/TMPRSS4 genes was highly associated with poor prognosis. Downregulation of TMPRSS4 significantly increased sensitivity to chemotherapy agents. In experiments using cisplatin, apoptosis and expression of the DNA-damage marker ¿-H2A was higher in cells lacking TMPRSS4. Moreover, in vivo assays demonstrated that tumors with no TMPRSS4 were significantly more sensitive to cisplatin than controls. These results show that TMPRSS4 can be considered as a novel target in NSCLC, whose inhibition increases chemosensitivity.
Autores: Exposito, F.; Villalba, M. ; Pajares, M. J.; et al.
ISSN 1556-0864  Vol. 14  Nº 10  2019  págs. S501
Autores: Idoate Gastearena, Miguel Ángel; Roca, P. G.; Benitez, E.; et al.
ISSN 0945-6317  Vol. 475  2019  págs. S32 - S32
Autores: Redin, E. ; Villalba, M. ; Exposito, F.; et al.
ISSN 1556-0864  Vol. 14  Nº 10  2019  págs. S698 - S699
Autores: De Andrea, Carlos Eduardo; Abengozar Muela, Marta; García Ros, David; et al.
ISSN 0023-6837  Vol. 99  Nº Supl. 1  2019  págs. 1820
Autores: Baraibar Argota, Iosune; Roman, M. ; López Erdozain, María Inés; et al.
ISSN 1556-0864  Vol. 14  Nº 10  2019  págs. S320 - S320
Autores: Salas Benito, Diego; De Andrea, Carlos Eduardo; Aramendía Beitia, José Manuel; et al.
ISSN 0923-7534  Vol. 30  Nº Supl. 5  2019  págs. 419
Autores: De Andrea, Carlos Eduardo; Villalba Esparza, María; Expósito Rincón, Francisco; et al.
ISSN 0023-6837  Vol. 99  Nº Supl. 1  2019 
Autores: De Andrea, Carlos Eduardo; Schalper, K. A. ; Fernández de Sanmamed Gutiérrez, Miguel; et al.
ISSN 1535-6108  Vol. 34  Nº 6  2018  págs. 876 - 878
Van den Eynde et al. publish in this issue of Cancer Cell that metastatic colorectal cancer shows marked heterogeneity in T cell infiltration among different lesions and patients. Measurements of T cell infiltration in metastases by immunoscore offer some prognostic information and support immune editing by coevolving adaptive immune responses.
Autores: Lozano Escario, María Dolores; Echeveste, José Ignacio; Abengozar Muela, Marta; et al.
ISSN 0003-9985  Vol. 142  Nº 3  2018  págs. 291 - 298
CONTEXT: - The rapid advances in targeted therapies in non-small cell lung cancer (NSCLC) make the optimization and implementation of cytology specimens for molecular testing a priority. Up to 70% of patients with NSCLC are diagnosed at advanced stages and tissue biopsies often cannot be taken. Although cytology samples provide high-quality material for molecular testing, molecular cytopathology is not yet well known or widely used. OBJECTIVE: - To report the many advances in molecular cytopathology and the suitability and utility of cytology samples in molecular and genetic testing of NSCLC. DATA SOURCES: - Data sources comprised published peer-reviewed literature and personal experience of the authors. CONCLUSIONS: - Molecular testing can be performed on cytologic specimens, especially on direct smears. Rapid on-site evaluation by cytopathologists has improved the adequacy and the management of cytology samples for molecular testing. Mutational profiling of NSCLC using next-generation sequencing can be performed on cytology samples from very small amounts of DNA. Fluorescence in situ hybridization assays on cytology specimens, including stained direct smear, offer some distinct advantages over their histologic counterpart, and are used to detect ALK and ROS1 rearrangements in NSCLC. Cytology specimens allow assessment of the entire tumor cell nucleus, avoiding signal loss from truncation artifacts. The use of cytology samples for assessing programmed death ligand-1 protein expression is currently being developed. Protocols for bisulfite conversion and DNA droplet digital polymerase chain reaction assays have been optimized for cytology smear to investigate aberrant DNA methylation of several NSCLC-related genes
Autores: Rodríguez Ruiz, María Esperanza; Pérez Gracia, José Luis; Rodríguez, I.; et al.
ISSN 0923-7534  Vol. 29  Nº 5  2018  págs. 1312 - 1319
Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-alpha and IFN-alpha. On days +8 and +10 of each cycle, patients received intratumoral image-guided 0.25mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m(2) was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre-and posttreatment PBMC from patients achieving durable stable disease (SD) were studied by IFNc ELISPOT-assays responding to tumor-lysate loaded DC and by TCR beta sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABR+ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-beta and IFN-alpha mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1 beta concentrations, especially in patients presenting SD. IFNc-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.
Autores: De Andrea, Carlos Eduardo; Abengozar Muela, Marta; Mejías Sosa, Luis Daniel; et al.
ISSN 0893-3952  Vol. 31  Nº Supl. 2  2018  págs. 158 - 158
Autores: Lozano Escario, María Dolores; Abengozar Muela, Marta; Labiano Miravalles, Tania; et al.
ISSN 0893-3952  Vol. 31  Nº Supl. 2  2018  págs. 158
Autores: Expósito Rincón, Francisco; Villalba, M.; Pajares Villandiego, María José; et al.
ISSN 1556-0864  Vol. 13  Nº 10  2018  págs. S521
Autores: De Andrea, Carlos Eduardo; San Julián Aranguren, Miguel; Bovée, J. V. M. G.;
ISSN 1875-9181  Vol. 10  Nº 3  2017  págs. 537 - 552
Cartilage-forming tumors of bone are a heterogeneous group of tumors with different molecular mechanisms involved. Enchondromas are benign hyaline cartilage-forming tumors of medullary bone caused by mutations in IDH1 or IDH2. Osteochondromas are benign cartilage-capped bony projections at the surface of bone. IDH mutations are also found in dedifferentiated and periosteal chondrosarcoma. A recurrent HEY1-NCOA2 fusion characterizes mesenchymal chondrosarcoma. Molecular changes are increasingly used to improve diagnostic accuracy in chondrosarcomas. Detection of IDH mutations or HEY1-NCOA2 fusions has already proved their immense value, especially on small biopsy specimens or in case of unusual presentation.
Autores: Malapelle, U.; Mayo-de-Las-Casas, C.; Molina-Vila, M. A.; et al.
ISSN 1934-662X  Vol. 125  Nº 8  2017  págs. 615 - 626
BACKGROUND: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit ¿ (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS: All laboratories using NGS (n¿=¿11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P¿=¿.171) and 5% (P¿=¿.063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n¿=¿2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures.
Autores: Rodríguez Ruiz, María Esperanza; Pérez Gracia, José Luis; Rodríguez González, Isabel; et al.
ISSN 0923-7534  Vol. 28  Nº Supl. 11  2017  págs. 48P
Autores: Villalba, M.; López, L.; Redrado Jordán, Miriam; et al.
ISSN 0213-3911  Vol. 32  Nº 9  2016  págs. 929 - 940
Metastatic spread is responsible for the majority of cancer deaths and identification of metastasis-related therapeutic targets is compulsory. TMPRSS4 is a pro-metastatic druggable transmembrane type II serine protease whose expression has been associated with the development of several cancer types and poor prognosis. To study the role and expression of this protease in cancer, we have developed molecular tools (active recombinant proteins and a polyclonal antibody) that can be used for diagnostic purposes and for testing anti-TMPRSS4 drugs. In addition, we have evaluated TMPRSS4 protein expression in several cancer tissue microarrays (TMAs). Full length and truncated TMPRSS4 recombinant proteins maintained the catalytic activity in two different expression systems (baculovirus and E. coli). Sensitivity of the rabbit polyclonal antisera against TMPRSS4 (ING-pAb) outperformed the antibody most commonly used in clinical settings. Analysis by immunohistochemistry in the different TMAs identified a subset of adenocarcinomas, squamous carcinomas, large cell carcinomas and carcinoids of the lung, which may define aggressive tumors. In conclusion, our biological tools will help the characterization of TMPRSS4 activity and protein expression, as well as the evaluation of anti-TMRSS4 drugs. Future studies should determine the clinical value of assessing TMPRSS4 levels in different types of lung cancer.
Autores: De Andrea, Carlos Eduardo; Zhu, J.-F.; Jin, H.; et al.
ISSN 0022-3417  Vol. 236  Nº 2  2015  págs. 210 - 218
Peripheral chondrosarcoma (PCS) develops as malignant transformation of an osteochondroma, a benign cartilaginous outgrowth at the bone surface. Its invasive, lobular growth despite low-grade histology suggests a loss of chondrocyte polarity. The known genetics of osteochondromagenesis include mosaic loss of EXT1 or EXT2 in both hereditary and non-hereditary cases. The most frequent genetic aberrations in human PCS also include disruptions of CDKN2A or TP53. In order to test the sufficiency of either of these to drive progression of an osteochondroma to PCS, we added conditional loss of Trp53 or Ink4a/Arf in an Ext1-driven mouse model of osteochondromagenesis. Each additional tumour suppressor silencing efficiently drove the development of growths that mimic human PCS. As in humans, lobules developed from both Ext1-null and Ext1-functional clones within osteochondromas. Assessment of their orientation revealed an absence of primary cilia in the majority of mouse PCS chondrocytes, which was corroborated in human PCSs. Loss of primary cilia may be responsible for the lost polarity phenotype ascribed to PCS. Cilia deficiency blocks proliferation in physeal chondrocytes, but cell cycle deregulation is sufficient to rescue chondrocyte proliferation following deciliation. This provides a basis of selective pressure for the frequent cell-cycle regulator silencing observed in peripheral chondrosarcomagenesis. Mosaic loss of Ext1 combined with loss of cell cycle regulators promotes peripheral chondrosarcomagenesis in the mouse and reveals deficient ciliogenesis in both the model and the human disease, explaining biological behaviour including lobular and invasive growth.
Autores: Salinas-Souza, C.; De Andrea, Carlos Eduardo; Bihl, M.; et al.
ISSN 0893-3952  Vol. 28  Nº 10  2015  págs. 1336 - 1342
Parosteal osteosarcoma, low-grade central osteosarcoma, and fibrous dysplasia share similar histological features that may pose a diagnostic challenge. The detection of GNAS mutations in primary bone tumors has been useful in clinical practice for diagnosing fibrous dysplasia. However, the recent report of GNAS mutations being detected in a significant proportion of parosteal osteosarcoma challenges the specificity of this mutation. As the number of cases reported in this study was small we set out to determine if these results could be reproduced. We studied 97 formalin-fixed paraffin-embedded low-grade osteosarcomas from 90 patients including 62 parosteal osteosarcomas, of which MDM2 amplification was detected in 79%, 11 periosteal osteosarcomas and 24 low-grade central osteosarcoma samples. The mutational status of GNAS was analyzed in codons p.R201, p.Q227, and other less common GNAS alterations by bidirectional Sanger sequencing and/or next generation sequencing using the Life Technologies Ion Torrent platform. GNAS mutations were not detected in any of the low-grade osteosarcomas from which informative DNA was extracted. Our findings therefore support prior observations that GNAS mutations are highly specific for fibrous dysplasia and occur rarely, if ever, in parosteal and other low-grade osteosarcomas.
Autores: De Andrea, Carlos Eduardo; Hogendoorn, P. C. W.;
ISSN 1756-2317  Vol. 20  Nº 5  2014  págs. 165 - 171
Chondroid tumours comprise a heterogeneous group of lesions with diverse morphological features and clinical behaviour. Over the past decade, a substantial number of chondroid tumours have been shown to harbour specific genetic abnormalities. These genetic alterations can be divided into two groups: tumours with specific translocations, and tumours with specific gene mutations. These genetic events give not only important insight in the tumourigenesis of chondroid tumours but offers new possibilities for molecular diagnosis. Some of these specific genetic changes have been implemented in routine pathology. This review aims to highlight the molecular genetics of chondroid tumours and their diagnostic applications.
Autores: Wiweger, M. I.; De Andrea, Carlos Eduardo; Scheepstra, K. W. F.; et al.
ISSN 1750-1172  Vol. 9  Nº 1  2014  págs. 35
Background Mutations in the EXT genes disrupt polymerisation of heparan sulphates (HS) and lead to the development of osteochondroma, an isolated/sporadic- or a multifocal/hereditary cartilaginous bone tumour. Zebrafish (Danio rerio) is a very powerful animal model which has shown to present the same cartilage phenotype that is commonly seen in mice model and patients with the rare hereditary syndrome, Multiple Osteochondroma (MO). Methods Zebrafish dackel (dak) mutant that carries a nonsense mutation in the ext2 gene was used in this study. A panel of molecular, morphological and biochemical analyses was used to assess at what step bone formation is affected and what mechanisms underlie changes in the bone formation in the ext2 mutant. Results During bone development in the ext2 -/- zebrafish, chondrocytes fail to undergo terminal differentiation; and pre-osteoblasts do not differentiate toward osteoblasts. This inadequate osteogenesis coincides with increased deposition of lipids/fats along/in the vessels and premature adipocyte differentiation as shown by biochemical and molecular markers. Also, the ext2-null fish have a muscle phenotype, i.e. muscles are shorter and thicker. These changes coexist with misshapen bones. Normal expression of runx2 together with impaired expression of osterix and its master regulator - xbp1 suggest that unfolded protein responses might play a role in MO pathogenesis. Conclusions Heparan sulphates are required for terminal differentiation of the cartilaginous template and consecutive formation of a scaffold that is needed for further bone development. HS are also needed for mesenchymal cell differentiation. At least one copy of ext2 is needed to maintain the balance between bone and fat lineages, but homozygous loss of the ext2 function leads to an imbalance between cartilage, bone and fat lineages. Normal expression of runx2 and impaired expression of osterix in the ext2 -/- fish indicate that HS are required by osteoblast precursors for their further differentiation towards osteoblastic lineage. Lower expression of xbp1, a master regulator of osterix, suggests that HS affect the `unfolded protein response¿, a pathway that is known to control bone formation and lipid metabolism. Our observations in the ext2-null fish might explain the musculoskeletal defects that are often observed in MO patients.
Autores: De Andrea, Carlos Eduardo; Hogendoorn, P. C. W.;
ISSN 0022-3417  Vol. 226  Nº 2  2012  págs. 219 - 228
Chondrocytes interact with their neighbours through their cartilaginous extracellular matrix (ECM). Chondrocyte¿matrix interactions compensate the lack of cell¿cell contact and are modulated by proteoglycans and other molecules. The epiphyseal growth plate is a highly organized tissue responsible for long bone elongation. The growth plate is regulated by gradients of morphogens that are established by proteoglycans. Morphogens diffuse across the ECM, creating short- and long-range signalling that lead to the formation of a polarized tissue. Mutations affecting genes that modulate cell¿matrix interactions are linked to several human disorders. Homozygous mutations of EXT1/EXT2 result in reduced synthesis and shortened heparan sulphate chains on both cell surface and matrix proteoglycans. This disrupts the diffusion gradients of morphogens and signal transduction in the epiphyseal growth plate, contributing to loss of cell polarity and osteochondroma formation. Osteochondromas are cartilage-capped bony projections arising from the metaphyses of endochondral bones adjacent to the growth plate. The osteochondroma cap is formed by cells with homozygous mutation of EXT1/EXT2 and committed stem cells/wild-type chondrocytes. Osteochondroma serves as a niche (a permissive environment), which facilitates the committed stem cells/wild-type chondrocytes to acquire secondary genetic changes to form a secondary peripheral chondrosarcoma. In such a scenario, the micro-environment is the site of the initiating processes that ultimately lead to cancer.
Autores: De Andrea, Carlos Eduardo; Kroon, H. M.; Wolterbeek, R.; et al.
ISSN 0893-3952  Vol. 25  Nº 9  2012  págs. 1275 - 1283
The distinction between benign and malignant cartilaginous tumors located peripherally in the bone may be a challenging task in surgical pathology. The aim of this study was to investigate interobserver reliability in histological diagnosis of cartilaginous tumors in the setting of multiple osteochondromas and to evaluate possible histological parameters that could differentiate among osteochondroma, low- and high-grade secondary peripheral chondrosarcoma. Interobserver reliability was assessed by 12 specialized bone-tumor pathologists in a set of 38 cases. Substantial agreement on diagnosis among all the reviewers was observed (intraclass correlation coefficient=0.78). Our study confirmed that mitotic figures and nuclear pleomorphism are hallmarks of high-grade secondary peripheral chondrosarcoma. However, despite the substantial agreement, we demonstrated that histology alone cannot distinguish osteochondroma from low-grade secondary peripheral chondrosarcoma in the setting of multiple osteochondromas, as nodularity, the presence of binucleated cells, irregular calcification, cystic/mucoid changes and necrosis were not helpful to indicate malignant transformation of an osteochondroma. On the other hand, among the concordant cases, the cartilage cap in osteochondroma was significantly less thicker than in low- and high-grade secondary peripheral chondrosarcoma. Therefore, our study showed that a multidisciplinary approach integrating clinical and radiographical features and the size of the cartilaginous cap in combination with a histological assessment are crucial to the diagnosis of cartilaginous tumors.
Autores: De Andrea, Carlos Eduardo; Reijnders, C. M. A.; Kroon, H. M.; et al.
ISSN 0950-9232  Vol. 31  Nº 9  2012  págs. 1095 - 1104
Secondary peripheral chondrosarcoma is the result of malignant transformation of a pre-existing osteochondroma, the most common benign bone tumor. Osteochondromas are caused by genetic abnormalities in EXT1 or EXT2: homozygous deletion of EXT1 characterizes sporadic osteochondromas (non-familial/solitary), and germline mutations in EXT1 or EXT2 combined with loss of heterozygosity define hereditary multiple osteochondromas. While cells with homozygous inactivation of EXT and wild-type cells shape osteochondromas, the cellular composition of secondary peripheral chondrosarcomas and the role of EXT in their formation have remained unclear. We report using a targeted-tiling-resolution oligo-array-CGH (array comparative genomic hybridization) that homozygous deletions of EXT1 or EXT2 are much less frequently detected (2/17, 12%) in sporadic secondary peripheral chondrosarcomas than expected based on the assumption that they originate in sporadic osteochondromas, in which homozygous inactivation of EXT1 is found in ~80% of our cases. FISH with an EXT1 probe confirmed that, unlike sporadic osteochondromas, cells from sporadic secondary peripheral chondrosarcomas predominantly retained one (hemizygous deleted loci) or both copies (wild-type) of the EXT1 locus. By immunohistochemistry, we confirm the presence of cells with dysfunctional EXT1 in sporadic osteochondromas and show cells with functional EXT1 in sporadic secondary peripheral chondrosarcomas. These immuno results were verified in osteochondromas and secondary peripheral chondrosarcomas in the setting of hereditary multiple osteochondromas. Our data therefore point to a model of oncogenesis in which the osteochondroma creates a niche in which wild-type cells with functional EXT are predisposed to acquire other mutations giving rise to secondary peripheral chondrosarcoma, indicating that EXT-independent mechanisms are involved in the pathogenesis of secondary peripheral chondrosarcoma.
Autores: De Andrea, Carlos Eduardo; Wiweger, M. I.; Bovée, J. V. M. G.; et al.
ISSN 0945-6317  Vol. 460  Nº 1  2012  págs. 95 - 102
Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours.
Autores: Waaijer, C. J. F.; De Andrea, Carlos Eduardo; Hamilton, A.; et al.
ISSN 0945-6317  Vol. 461  Nº 4  2012  págs. 475 - 481
Chondrosarcomas are malignant cartilage-forming tumours that can arise centrally (in the medulla) or peripherally (at the surface) of the bone. They are classified into three histological grades which correspond to the clinical severity. Previous studies by our group have shown altered signal transduction of the fibroblast growth factor and Wnt signalling pathways during peripheral chondrosarcoma progression. Heparan sulphate (HS) is a glycosaminoglycan that facilitates receptor binding of multiple growth factors, in which the sulphation of 6O position plays a pivotal role. 6O-Sulphation occurs through three HS 6O-sulphotransferases (HS6ST1-3) and is fine-tuned by two endosulphatases (SULF1-2) that remove 6O-sulphate groups. We have investigated whether the expression of HS6STs and SULFs changes during chondrosarcoma progression and have determined 6O-sulphation levels in two chondrosarcoma cell lines. Immunohistochemistry on tissue microarrays of chondrosarcomas showed that HS6ST3 and SULF1 were highly expressed in most chondrosarcomas, whereas SULF2 expression was absent in most cases. HS6ST1 and HS6ST2 expression are significantly increased during chondrosarcoma progression, which suggest that 6O-sulphation is increased during progression. This was confirmed in one grade III chondrosarcoma cell line, which showed a dramatically increased 6O-sulphation compared to an articular chondrocyte cell line by HPLC; another cell line showed an increased expression of one 6O-sulphated HS disaccharide. In conclusion, our results show increased HS6ST1 and HS6ST2 expression during chondrosarcoma progression and increased HS 6O-sulphation in vitro. As 6O-sulphation plays an important role in signal transduction, altered HS6STexpression might be associatedwith changes in signal transduction pathways in chondrosarcoma progression.
Autores: Mohseny, A. B.; Cai, Y.; Kuijjer, M.; et al.
ISSN 0959-8049  Vol. 48  Nº 18  2012  págs. 3429 - 3438
High-grade conventional osteosarcoma is a malignant tumour predominantly affecting adolescents and, despite multimodal intensive therapy, lethal for one third of the patients. Although there is currently detailed knowledge of normal skeletal development, this has not been integrated into research on the genesis of osteosarcoma. Recently we showed that the canonical Wnt pathway is not active in osteosarcoma and that its reactivation is disadvantageous to osteosarcoma cells. Since Wnt is regulating normal skeletogenesis together with other pathways, here we report on the activities of the bone morphogenic protein (BMP), the transforming growth factor beta (TGFß) and the hedgehog (Hh) pathways in osteosarcoma. Human osteosarcoma samples (n = 210), benign bone tumours of osteoblastic lineage called osteoblastoma (n = 25) and osteosarcoma cell lines (n = 19) were examined. For pathway activity luciferase transcriptional reporter assays and gene and protein expression analyses were performed. Immunohistochemical analysis of phosphorylated Smad1 and Smad2, the intracellular effectors of BMP and TGFß, respectively, showed nuclear expression of both proteins in 70% of the osteosarcoma samples at levels comparable to osteoblastoma. Interestingly cases with lower expression showed significantly worse disease free survival. This may imply that drugs restoring impaired signalling pathways in osteosarcoma might change the tumour¿s aggressive clinical course, however targeted pathway modulation in vitro did not affect cell proliferation.
Autores: De Andrea, Carlos Eduardo; Prins, F. A.; Wiweger, M.; et al.
ISSN 0022-3417  Vol. 224  Nº 2  2011  págs. 160 - 168
Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine¿PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (ß3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.
Autores: De Andrea, Carlos Eduardo; Petrilli, A. S.; Jesus-Garcia, R.; et al.
ISSN 1936-2625  Vol. 4  Nº 2  2011  págs. 169 - 174
Osteosarcoma is the most frequent primary malignant bone tumor. Distinct histological features are distinguishable based on the morphology of the tumor. Differences in nuclei size and shape are often observed in osteosarcoma reflecting its broad histopathological heterogeneity. This study explores the relevance of two nuclear parameters in osteosarcoma: large area and round shape. Computerized nuclear morphometry was performed in 56 conventional osteosarcoma preoperative biopsies. The mean patient follow-up time was 35.1 months. Based on the nuclear area, no significant difference (P = 0.09) in overall survival between patients with large (¿ 42.5 ¿m2) and small (< 42.5 ¿m2) tumor nuclei was found. However, when cases with large and round nuclei were analyzed jointly (¿ 42.5 ¿m2 and coefficient of nuclear roundness ¿ 0.7), these two parameters together were likely to be a predictive factor (P = 0.05). Osteosarcoma patients with large and round tumor nuclei had a better outcome than patients with small and polymorphic (ovoid or spindle-shaped) nuclei. In this study, nuclear morphometry proved to be a useful tool to shed light on the biology of osteosarcoma showing that some morphometric parameters can be easily applied to help identifying patients with a good prognosis.
Autores: Wiweger, M. I.; Avramut, C. M.; De Andrea, Carlos Eduardo; et al.
ISSN 0022-3417  Vol. 223  Nº 4  2011  págs. 531 - 542
Proteoglycans are molecules consisting of protein cores onto which sugar chains, i.e., glycosaminoglycans (GAGs) such as heparan or chondroitin sulphates, are attached. Proteoglycans are produced by nearly all cells, and once secreted they become a major component of the extracellular matrix. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of GAGs have been found in osteochondromas and osteoarthritis. The zebrafish (Danio rerio) exhibits fast development, a growth plate-like organization of its craniofacial skeleton and an availability of various mutants, making it a powerful model for the study of human skeletal disorders with unknown aetiology. We analysed skeletons from five zebrafish lines with known mutations in genes involved in proteoglycan synthesis: dackel (dak/ext2), lacking heparan sulphate; hi307 (ß3gat3), deficient for most GAGs; pinscher (pic/slc35b2), presenting defective sulphation of GAGs and other molecules; hi954 (uxs1), lacking Notch and most GAGs due to impaired protein xylosylation; and knypek (kny/gpc4), missing the protein core of the Glypican-4 proteoglycan. Here we show that each mutant displays different phenotypes related to: (a) cartilage morphology; (b) composition of the extracellular matrix; (c) ultrastructure of the extracellular matrix; and (d) the intracellular ultrastructure of chondrocytes, proving that sulphated GAGs orchestrate the cartilage intra- and extracellular ultrastructures. The mild phenotype of the hi307 mutant suggests that proteoglycans consisting of a protein core and a short sugar linker might suffice for proper chondrocyte stacking. Finally, knypek supports the involvement of Glypican-4 in the craniofacial phenotype of Simpson¿Golabi¿Behmel syndrome and suggests GPC4 as a modulator of the overgrowth phenotype that is associated with this syndrome and is primarily caused by a mutation in GPC3. Moreover, we speculate on the potential involvement of SLC35B2, ß3GAT3 and UXS1 in skeletal dysplasias. This work promotes the use of zebrafish as a model of human skeletal development and associated pathologies.
Autores: Mohseny, A. B.; Tieken, C.; van der Velden, P. A; et al.
ISSN 1045-2257  Vol. 49  Nº 12  2010  págs. 1095 - 1103
Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14ARF function.
Autores: De Andrea, Carlos Eduardo; Wiweger, M.; Prins, F.; et al.
ISSN 1530-0307  Vol. 90  Nº 7  2010  págs. 1091 - 1101
Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture (`mosaic¿) of normal lining (EXT+/¿ or EXTwt/wt) and EXT¿/¿ cells in the cartilaginous cap of osteochondromas.
Autores: Hogendoorn, P. C. W.; De Andrea, Carlos Eduardo
Libro:  Encyclopedia of Cancer
2019  págs. 153 - 169
Bone tumors are considered difficult to diagnose by most pathologists as they are rare, have overlapping morphology, need radiological correlation, and the usefulness of immunohistochemistry is limited to a relative small number of differential diagnosis, making conventional morphology and knowledge of clinicoradiological presentation the cornerstones of the diagnosis. Three main groups of bone tumors are identified: (i) tumors with specific translocations combined with a relatively simple karyotype involving chromosomal translocations (Ewing sarcoma, aneurysmal bone cyst, mesenchymal chondrosarcoma), (ii) tumors with specific gene mutations or amplifications or deletions (chondrosarcoma, giant cell tumor of bone, chondroblastoma, fibrous dysplasia, chordoma osteochondroma), and (iii) sarcomas with genetic instability and as a consequence complex karyotypes (osteosarcoma). An accurate diagnosis is essential to guide the surgeons hands, predict clinical outcome and for multimodal treatment decision-making.
Autores: De Andrea, Carlos Eduardo; Hogendoorn, P. C. W.; Bovée, J. V. M. G. ;
Libro:  eLS
2016  págs. -
Autores: Bovée, J. V. M. G. ; De Andrea, Carlos Eduardo
Título: Bone tumors
Libro:  Sternberg's diagnostic surgical pathology
2015  págs. 273 - 316
Autores: Montuenga Badía, Luis; Bodegas Frías, María Elena; De Andrea, Carlos Eduardo; et al.
Libro:  Técnicas en histología y biología celular
2014  págs. 61 - 84
Autores: Montuenga Badía, Luis; Bodegas Frías, María Elena; De Andrea, Carlos Eduardo; et al.
Libro:  Técnicas en histología y biología celular
2014  págs. 35 - 60
Autores: De Andrea, Carlos Eduardo
Chondrocytes interact with their neighbours through their cartilaginous extracellular matrix (ECM). Chondrocyte¿matrix interactions compensate the lack of cell¿cell contact and are modulated by proteoglycans and other molecules. The epiphyseal growth plate is a highly organized tissue responsible for long bone elongation. The growth plate is regulated by gradients of morphogens that are established by proteoglycans. Morphogens diffuse across the ECM, creating short- and long-range signalling that lead to the formation of a polarized tissue. Mutations affecting genes that modulate cell¿matrix interactions are linked to several human disorders. Homozygous mutations of EXT1/EXT2 result in reduced synthesis and shortened heparan sulphate chains on both cell surface and matrix proteoglycans. This disrupts the diffusion gradients of morphogens and signal transduction in the epiphyseal growth plate, contributing to loss of cell polarity and osteochondroma formation. Osteochondromas are cartilage-capped bony projections arising from the metaphyses of endochondral bones adjacent to the growth plate. The osteochondroma cap is formed by cells with homozygous mutation of EXT1/EXT2 and committed stem cells/wild type chondrocytes. Osteochondroma serves as a niche (a permissive environment), which facilitates the committed stem cells/wild-type chondrocytes to acquire secondary genetic changes to form a secondary peripheral chondrosarcoma. In such a scenario, the microenvironment is the site of the initiating processes that ultimately lead to cancer.