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1.  Mutations of epigenetic regulatory genes are common in thymic carcinomas 
Scientific Reports  2014;4:7336.
Genetic alterations and etiology of thymic epithelial tumors (TETs) are largely unknown, hampering the development of effective targeted therapies for patients with TETs. Here TETs of advanced-stage patients enrolled in a clinical trial of molecularly-guided targeted therapies were employed for targeted sequencing of 197 cancer-associated genes. Comparative sequence analysis of 78 TET/blood paired samples obtained from 47 thymic carcinoma (TC) and 31 thymoma patients revealed a total of 86 somatic non-synonymous sequence variations across 39 different genes in 33 (42%) TETs. TCs (62%; 29/47) showed higher incidence of somatic non-synonymous mutations than thymomas (13%; 4/31; p < 0.0001). TP53 was the most frequently mutated gene in TETs (n = 13; 17%), especially in TCs (26%), and was associated with a poorer overall survival (p < 0.0001). Genes in histone modification [BAP1 (n = 6; 13%), SETD2 (n = 5; 11%), ASXL1 (n = 2; 4%)], chromatin remodeling [SMARCA4 (n = 2; 4%)], and DNA methylation [DNMT3A (n = 3; 7%), TET2 (n = 2; 4%), WT1 (n = 2; 4%)] pathways were recurrently mutated in TCs, but not in thymomas. Our results suggest a potential disruption of epigenetic homeostasis in TCs, and a substantial difference in genetic makeup between TCs and thymomas. Further investigation is warranted into the roles of epigenetic dysregulation in TC development and its potential for targeted therapy.
PMCID: PMC4258655  PMID: 25482724
2.  Copy number aberrations of genes regulating normal thymus development in thymic epithelial tumors 
To determine if the deregulation of genes relevant for normal thymus development can contribute to the biology of thymic epithelial tumors.
Experimental Design
Using array comparative genomic hybridization, we evaluated the copy number aberrations of genes regulating thymus development. The expression of genes most commonly involved in copy number aberrations was evaluated by immunohistochemistry and correlated with patients' outcome. Correlation between FOXC1 copy number loss and gene expression was determined in a confirmation cohort. Cell lines were used to test the role of FOXC1 in tumors.
Among 31 thymus development-related genes, PBX1 copy number gain and FOXC1 copy number loss were presented in 43.0% and 39.5% of the tumors respectively. Immunohistochemistry on a series of 132 thymic epithelial tumors including those evaluated by comparative genomic hybridization, revealed a correlation between protein expression and copy number status only for FOXC1 but not for PBX1. Patients with FOXC1–negative tumors had a shorter time to progression and a trend for a shorter disease related survival. The correlation between FOXC1 copy number loss and mRNA expression was confirmed in a separate cohort of 27 thymic epithelial tumors. Ectopic FOXC1 expression attenuated anchorage-independent cell growth and cell migration in vitro.
Our data support a tumor suppressor role of FOXC1 in thymic epithelial tumors.
PMCID: PMC3630263  PMID: 23444221
thymic epithelial tumors; thymoma; thymic carcinoma; FOXC1; PBX1; thymus development
3.  Are MSCs angiogenic cells? New insights on human nestin-positive bone marrow-derived multipotent cells 
Recent investigations have made considerable progress in the understanding of tissue regeneration driven by mesenchymal stromal cells (MSCs). Data indicate the anatomical location of MSC as residing in the “perivascular” space of blood vessels dispersed across the whole body. This histological localization suggests that MSCs contribute to the formation of new blood vessels in vivo. Indeed, MSCs can release angiogenic factors and protease to facilitate blood vessel formation and in vitro are able to promote/support angiogenesis. However, the direct differentiation of MCSs into endothelial cells is still matter of debate. Most of the conflicting data might arise from the presence of multiple subtypes of cells with heterogeneous morpho functional features within the MSC cultures. According to this scenario, we hypothesize that the presence of the recently described Mesodermal Progenitor Cells (MPCs) within the MSCs cultures is responsible for their variable angiogenic potential. Indeed, MPCs are Nestin-positive CD31-positive cells exhibiting angiogenic potential that differentiate in MSC upon proper stimuli. The ISCT criteria do not account for the presence of MPC within MSC culture generating confusion in the interpretation of MSC angiogenic potential. In conclusion, the discovery of MPC gives new insight in defining MSC ancestors in human bone marrow, and indicates the tunica intima as a further, and previously overlooked, possible additional source of MSC.
PMCID: PMC4207020  PMID: 25364727
mesenchymal stromal cells; endothelial differentiation; angiogenesis; adult stem cells; bone marrow; nestin; neo-vascolarization; in vivo MSC
4.  Whole Genome and Transcriptome Sequencing of a B3 Thymoma 
PLoS ONE  2013;8(4):e60572.
Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomic hybridization using Agilent platform, transcriptome sequencing using HiSeq 2000 (Illumina) and whole genome sequencing using Complete Genomics Inc platform. Whole genome sequencing determined, in tumor and normal, the sequence of both alleles in more than 95% of the reference genome (NCBI Build 37). Copy number (CN) aberrations were comparable with those previously described for B3 thymomas, with CN gain of chromosome 1q, 5, 7 and X and CN loss of 3p, 6, 11q42.2-qter and q13. One translocation t(11;X) was identified by whole genome sequencing and confirmed by PCR and Sanger sequencing. Ten single nucleotide variations (SNVs) and 2 insertion/deletions (INDELs) were identified; these mutations resulted in non-synonymous amino acid changes or affected splicing sites. The lack of common cancer-associated mutations in this patient suggests that thymomas may evolve through mechanisms distinctive from other tumor types, and supports the rationale for additional high-throughput sequencing screens to better understand the somatic genetic architecture of thymoma.
PMCID: PMC3618227  PMID: 23577124
5.  NUT Rearrangement is Uncommon in Human Thymic Epithelial Tumors 
Journal of Thoracic Oncology  2012;7(4):744-750.
Thymic carcinomas represent the most aggressive histotype of thymic epithelial tumors (TETs). The 2004 World Health Organization (WHO) classification has assigned a subgroup of thymic carcinomas as t(15:19) carcinomas based on the presence of t(15;19), a translocation found in poorly differentiated and highly aggressive NUT midline carcinomas (NMC). These tumors are characterized by rearrangement of the NUT (nuclear protein in testis) gene on chromosome 15q14, which in most cases fuses to the bromomdomain containing 4 (BRD4) gene on chromosome 19p13.1 through reciprocal t(15;19) translocation, resulting in constitutive BRD4-NUT fusion protein expression. To our knowledge, NUT translocation has been reported only in four thymic carcinomas. Due to the rarity of TETs, the prevalence of NUT rearrangement in TETs has however never been systematically explored.
Formalin-fixed paraffin-embedded samples of histologically-confirmed TETs were evaluated for NUT expression and rearrangement by immunohistochemistry and fluorescence in-situ hybridization respectively.
A series of 148 TETs (37 carcinomas and 111 thymomas) were examined for NUT expression and rearrangement. Only one thymic carcinoma (2.7% of thymic carcinomas or 0.68% of TETs) was found positive for NUT expression and rearrangement.
Rearrangement of NUT is infrequent in TETs. We propose that caution should be taken to distinguish t(15;19) thymic carcinoma from other mediastinal carcinomas, as NMCs are often associated with dreadful prognosis or overt lethality.
PMCID: PMC3308127  PMID: 22425924
t(15;19) translocation; BRD4-NUT; thymoma; thymic carcinoma
6.  Loss of 18q22.3 involving the carboxypeptidase of glutamate-like gene is associated with poor prognosis in resected pancreatic cancer 
Clinical Cancer Research  2011;18(2):524-533.
Pancreatic cancer is the 4th-leading cause of cancer-related death, and studies on the clinical relevance of its genomic imbalances are warranted.
Experimental Design
Recurrent copy number alterations of cytobands and genes were analyzed by array comparative genomic hybridization (aCGH) in 44 resected pancreatic cancer specimens. Prognostic markers identified by aCGH were validated by PCR gene copy number assay in an independent validation cohort of 61 resected pancreatic cancers. The functions of gene identified were evaluated by proliferation, cell cycle and migration assays in pancreatic cancer cells.
We demonstrated recurrent copy number gains and losses in the first cohort. Loss of 18q22.3 was significantly associated with short-term overall survival in the first cohort (p=0.019). This cytoband includes the Carboxypeptidase of glutamate-like (CPGL) gene. CPGL gene deletion was associated with shorter overall survival in the validation cohort (p=0.003). CPGL deletion and mutations of TP53 or Kras appear to be independent events. A Cox model analysis of the two cohorts combined showed that loss of 18q22.3/deletion of the CPGL gene was an independent poor prognostic factor for overall survival (hazard ratio=2.72, p=0.0007). Reconstitution of CPGL or its splicing-variant CPGL-B into CPGL-negative pancreatic cancer cells attenuated cell growth, migration, and induced G1-accumulation.
Loss of 18q22.3/deletion of the CPGL gene is a poor prognostic marker in resected pancreatic cancer, and functional studies suggest the CPGL gene as growth suppressor gene in pancreatic cancer.
PMCID: PMC3261299  PMID: 22128300
Pancreatic cancer; Comparative genomics; CPGL protein; human; Prognosis; Growth suppressor
7.  Thymic Malignancies: From Clinical Management to Targeted Therapies 
Journal of Clinical Oncology  2011;29(36):4820-4827.
A key challenge in the treatment of thymoma and thymic carcinoma (TC) is in improving our understanding of the molecular biology of these relatively rare tumors. In recent years, significant efforts have been made to dissect the molecular pathways involved in their carcinogenesis. Here we discuss the results of large-scale genomic analyses conducted to date and review the most active chemotherapies and targeted treatments.
We reviewed the literature for chemotherapeutic trials in the last 20 years and trials involving targeted therapies between 1999 and 2010. The search was supplemented by a review of abstracts presented at the annual meetings of the American Society of Clinical Oncology (from 1999 to 2010), at the first International Conference on Thymic Malignancies in 2009, and at a follow-up meeting of the newly formed International Thymic Malignancies Interest Group in 2010.
Surgery remains the treatment of choice for operable tumors, whereas chemotherapy is standard in locally advanced and metastatic disease. Thus far, targeted therapies have been developed empirically. Histone deacetylase inhibitors have shown some activity in thymoma whereas sunitinib may be active in TC. There are no data to support the use of HER2- or EGFR-targeted therapies in thymic malignancies.
Drug development for the treatment of thymic malignancies is difficult because of the rarity of these tumors. Ethnic differences are becoming apparent, with aggressive subtypes being observed in Asians and African Americans. Incremental improvements in our understanding of tumor biology suggest that molecular profiling–directed therapies may be the preferred route of investigation in the future.
PMCID: PMC3675690  PMID: 22105817
8.  Arsenic trioxide and ascorbic acid interfere with the BCL2 family genes in patients with myelodysplastic syndromes: an ex-vivo study 
Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients’ bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28.
Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan® Assays in 384-Well Microfluidic Cards “TaqMan® Human Apoptosis Array”.
Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH).
ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were up-regulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases.
These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.
PMCID: PMC3465246  PMID: 22964015
ATO; Ascorbic acid; Myelodysplastic syndromes; MDS; Apoptosis
9.  MicroRNA expression and clinical outcomes in patients treated with adjuvant chemotherapy after complete resection of non-small cell lung carcinoma 
Cancer research  2010;70(21):8288-8298.
This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non-small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155 and let-7a was determined by quantitative real-time PCR in paraffin embedded formalin fixed tumor specimens from 639 IALT patients. Prognostic and predictive value of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathological prognostic factors and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed association of TP53 mutation status and miR-34a/b/c expression, EGFR and KRAS mutation status and miR-21 and Let-7a expression, respectively. Finally, association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 where a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the miRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort of radically resected NSCLC randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only.
PMCID: PMC2970724  PMID: 20978195
non–small cell lung cancer; adjuvant chemotherapy; randomized trial; biomarker; drug resistance; microRNA
10.  IGF-1R and p-AKT expression in 132 resected thymomas and thymic carcinomas 
Cancer  2010;116(20):4686-4695.
Thymic malignancies are rare tumors. The IGF-1/IGF-1R system is involved in the development of the thymus. IGF-1R expression in thymic epithelial malignancies is unknown.
We investigated the expression of insulin-like growth factor 1 receptor (IGF-1R) and phospho-AKT Ser-473 (p-AKT) by immunohistochemistry and the clinicopathological correlations in a retrospective single institution surgical series of 132 thymic epithelial malignancies.
Earlier stages, less aggressive histological types, and complete resections were significant positive prognostic factors for disease-related survival and progression-free survival, and female gender was a better prognostic factor for disease-related survival. IGF-1R and p-AKT proteins were expressed in 20% and 36% of thymic tumors, respectively. They were both more commonly expressed in relapsed than in primary tumors, in more aggressive subtypes and more advanced stages. There was a trend for better survival and progression-free survival in IGF-1R or p-AKT expression negative cases in the whole series; considering the 91 primary tumors only, IGF1R expression was associated with worse progression-free survival (p<0.001).
This retrospective analysis demonstrates stage, histology, gender and resection type as major prognostic factors. The expression of IGF-1R and p-AKT in thymic tumors suggests IGF-1R as a potential target for treatment.
PMCID: PMC3057766  PMID: 20597130
thymoma; IGF-1R; expression; immunohistochemistry; prognosis

Results 1-10 (10)