Konstantin Agelopoulos

Cytogenetic Alterations and Cytokeratin Expression Patterns in Breast Cancer: Integrating a New Model of Breast Differentiation into Cytogenetic Pathways of Breast Carcinogenesis

The introduction of a concept proposing multiple cellular subgroups in the normal female breast, including cytokeratin 5/6 (Ck 5/6)-positive progenitor cells, offers a new explanation for the existence of highly aggressive breast cancers with and without Ck 5/6 expression. Using the tissue microarray technique, 166 breast cancer cases, all characterized by comparative genomic hybridization, were evaluated by immunohistochemistry, using 15 different antibodies (estrogen receptor, progesterone receptor, p53, Ki-67, c-erbB2, epidermal growth factor receptor, cyclins A, D1, and E, bcl-2, p21, p27, Ck 5/6, Ck 8/18, and smooth muscle actin) and chromogenic in situ hybridization for c-erbB2. Biomathematical cluster analysis was applied to confirm the conventional interpretation of the results by an independent approach. Ck 5/6-positive breast carcinomas were in general negative for estrogen receptor and progesterone receptor, were highly proliferating (as reflected by Ki67 and cyclin A), and were associated with specific protein expression patterns, such as expression of p53 and epithelial growth factor receptor (all related to more aggressive tumor behavior), which could further be demonstrated by biomathematical cluster analysis. In contrast Ck 5/6-negative breast carcinomas revealed a lower tumor proliferation rate, an increased expression of p21, p27, c-erbB2, and bcl-2, and a significantly lower number of genetic alterations, with losses of chromosomal material of 16q as the most common genetic alteration. Our data give the first hints to the hypothesis that different cellular subgroups in the female breast give rise to subgroups of breast carcinomas with differing protein expression and cytogenetic alteration patterns that may be related to clinical behavior.

Tumor-Infiltrating Macrophages Are Associated with Metastasis Suppression in High-Grade Osteosarcoma: A Rationale for Treatment with Macrophage Activating Agents

Purpose: High-grade osteosarcoma is a malignant primary bone tumor with a peak incidence in adolescence. Overall survival (OS) of patients with resectable metastatic disease is approximately 20%. The exact mechanisms of development of metastases in osteosarcoma remain unclear. Most studies focus on tumor cells, but it is increasingly evident that stroma plays an important role in tumorigenesis and metastasis. We investigated the development of metastasis by studying tumor cells and their stromal context. Experimental Design: To identify gene signatures playing a role in metastasis, we carried out genome-wide gene expression profiling on prechemotherapy biopsies of patients who did (n = 34) and patients who did not (n = 19) develop metastases within 5 years. Immunohistochemistry (IHC) was performed on pretreatment biopsies from 2 additional cohorts (n = 63 and n = 16) and corresponding postchemotherapy resections and metastases. Results: A total of 118/132 differentially expressed genes were upregulated in patients without metastases. Remarkably, almost half of these upregulated genes had immunological functions, particularly related to macrophages. Macrophage-associated genes were expressed by infiltrating cells and not by osteosarcoma cells. Tumor-associated macrophages (TAM) were quantified with IHC and associated with significantly better overall survival (OS) in the additional patient cohorts. Osteosarcoma samples contained both M1- (CD14/HLA-DRα positive) and M2-type TAMs (CD14/CD163 positive and association with angiogenesis). Conclusions: In contrast to most other tumor types, TAMs are associated with reduced metastasis and improved survival in high-grade osteosarcoma. This study provides a biological rationale for the adjuvant treatment of high-grade osteosarcoma patients with macrophage activating agents, such as muramyl tripeptide. Clin Cancer Res; 17(8); 2110–9. ©2011 AACR.

Mechanisms of egfr Gene Transcription Modulation: Relationship to Cancer Risk and Therapy Response

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. For predictive cancer diagnostics and therapeutic targeting of EGFR, it is important to know how the expression level of EGFR is controlled and related to receptor signaling. A novel transcriptional regulation mechanism has been described that depends on the length of a CA repeat in intron 1 [CA simple sequence repeat 1 (CA SSR I)] of the EGFR gene. Thereby, the number of CA repeats is inversely correlated to pre-mRNA synthesis. Indirect evidence for the importance of this mechanism includes the preferential occurrence of amplifications in cancer tissue harboring short CA repeats in this sequence and the discovery of distinct alleles in young breast cancer patients with a family history of the disease and in Japanese breast cancer patients. It can be postulated that the length of the CA repeat influences DNA bendability and, in consequence, the binding of repressor proteins. In summary, it seems that the CA SSR I represents an inherited variable for response to anti-EGFR therapies that could be determined before therapy. Moreover, the potential for synergistic effects with other polymorphism [e.g., EGFR R497K (HER-1 497K) and CCND1 A870G] leading to a simultaneous increase of EGFR signaling activity and expression should be investigated. From a practical perspective, assessment of the CA SSR I number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it is a constant feature that does not change over time and can be easily measured in normal and cancer tissues (blood cells, skin, and tumor biopsies) in an assay that is technically simple, objective, and even quantitative.

Genome wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane

BackgroundThe chick chorio-allantoic membrane (CAM) assay is a commonly used method for studying angiogenic or anti-angiogenic activities in vivo. The ease of access allows direct monitoring of tumour growth by biomicroscopy and the possibility to screen many samples in an inexpensive way. The CAM model provides a powerful tool to study effects of molecules, which interfere with physiological angiogenesis, or experimental tumours derived from cancer cell lines. We therefore screened eight osteosarcoma cell lines for their ability to form vascularized tumours on the CAM.FindingsWe implanted 3-5 million cells of human osteosarcoma lines (HOS, MG63, MNNG-HOS, OST, SAOS, SJSA1, U2OS, ZK58) on the CAM at day 10 of embryonic development. Tumour growth was monitored by in vivo biomicroscopy at different time points and tumours were fixed in paraformaldehyde seven days after cell grafting. The tissue was observed, photographed and selected cases were further analyzed using standard histology.From the eight cell lines the MNNG-HOS, U2OS and SAOS were able to form solid tumours when grafted on the CAM. The MNNG-HOS tumours showed the most reliable and consistent growth and were able to penetrate the chorionic epithelium, grow in the CAM stroma and induce a strong angiogenic response.ConclusionsOur results show that the CAM assay is a useful tool for studying osteosarcoma growth. The model provides an excellent alternative to current rodent models and could serve as a preclinical screening assay for anticancer molecules. It might increase the speed and efficacy of the development of new drugs for the treatment of osteosarcoma.

Adenosine A2A receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder

Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A2A receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A2A receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

A short-term in vivo model for giant cell tumor of bone

BackgroundBecause of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.MethodsFresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH).ResultsThe suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.ConclusionsA reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.

Epidermal Growth Factor Receptor Expression in High-Grade Osteosarcomas Is Associated with a Good Clinical Outcome

Purpose: The expression of the epidermal growth factor receptor (EGFR) in osteosarcomas has repeatedly been described. With the introduction of anti-EGFR–targeted therapies in clinical practice, these findings regain increased attention. Experience with anti-EGFR–targeted therapies in other cancers has made clear that besides the expression status of EGFR, a detailed knowledge about gene mutations is of major predictive power. We therefore aimed to explore the EGFR expression and gene mutation status in high-grade osteosarcomas. Experimental Design: We investigated tumor samples of osteosarcoma patients of all age groups by means of immunohistochemistry (n = 111) and egfr fluorescence in situ hybridization (n = 39). Sixty-three patients were treated according to the Cooperative Osteosarcoma Study Group protocols and complete clinical follow-up was available in these cases. Results: Ninety-one of 111 (81%) of osteosarcomas revealed an expression of EGFR. EGFR expression showed a dose-response relation with improved event-free and overall survival. This was independent of the degree of tumor regression due to neoadjuvant chemotherapy. Nine of 39 (23%) osteosarcomas showed egfr amplifications by means of fluorescence in situ hybridization. All these cases expressed EGFR. When comparing EGFR expression between primary biopsy and resection specimen (n = 19), viable residual tumor cells in resection specimens revealed a lower EGFR expression and a tendency toward membranous staining compared with the initial biopsy. Conclusions: In conclusion, expression and amplification of EGFR are frequently observed in high-grade osteosarcomas and are associated with improved prognosis in a dose-responsive way. This implies that low EGFR expression possibly predicts lack of response to conventional treatment in high-grade osteosarcomas and may warrant a more intensive therapeutic approach, although not based on EGFR targeting.

Deep sequencing in conjunction with expression and functional analyses reveals activation of FGFR1 in Ewing sarcoma.

Purpose: A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. Experimental Design: Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas. Results: Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity. Conclusions: FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma. Clin Cancer Res; 21(21); 4935–46. ©2015 AACR.

Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468

BackgroundIncreased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.MethodsThe basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.ResultsLow and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.ConclusionProgressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.