Iva Zambo

Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

BackgroundNestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines.MethodsUsing immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines.ResultsNestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+.ConclusionOur results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.

CD133 Expression and Identification of CD133/nestin Positive Cells in Rhabdomyosarcomas and Rhabdomyosarcoma Cell Lines

Background: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. Methods: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. Results: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. Conclusion: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.

Nestin expression in high-grade osteosarcomas and its clinical significance

Nestin has been detected in various malignancies and its expression correlates with advanced grade in some neoplasms. The aim of this study was to examine nestin expression in high-grade osteosarcomas and to determine its prognostic value. Using immunohistochemistry and immunofluorescence, we evaluated nestin expression in tumor tissue samples from 45 patients with high-grade osteosarcomas. In both methods, the frequency of nestin-positive tumor cells was classified into three categories (1+, 2+ and 3+ for immunohistochemistry; 1F+, 2F+ and 3F+ for immunofluorescence) and clinicopathological correlations were statistically evaluated and analyzed. Nestin-positive tumor cells were detected in all of the examined osteosarcomas using both immunohistochemistry and immunofluorescence, although the proportion of undoubtedly positive neoplastic cells varied in individual samples from a few nestin-positive tumor cells to diffuse nestin positivity. High levels of nestin expression detected by immunofluorescence (2F+ and 3F+) were associated with worse clinical outcomes (OS, p=0.031; EFS, p<0.001). However, high levels of nestin expression as measured by immunohistochemistry trended towards shorter patient survival rates but did not reach statistical significance. Despite significantly shorter survival rates observed in patients with high levels of nestin expression assessed by immunofluorescence, nestin does not seem to represent a powerful prognostic marker that would be superior to conventional methods.

Cancer stem cell markers in pediatric sarcomas: Sox2 is associated with tumorigenicity in immunodeficient mice

The three most frequent pediatric sarcomas, i.e., Ewing’s sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas—ABCG2, CD133, and nestin—was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.

Expression of nestin, CD133 and ABCG2 in relation to the clinical outcome in pediatric sarcomas.

BACKGROUND Nestin, CD133 and ABCG2 are recently discussed as putative markers, co-expression of which might determine a cancer stem cell (CSC) phenotype in sarcomas. OBJECTIVE Our study is focused on immunohistochemical analysis of nestin, CD133 and ABCG2 expression in rhabdomyosarcoma, Ewing sarcoma and osteosarcoma. Furthermore, we also analyzed the possible correlation of nestin, CD133 and ABCG2 expression levels with the patient outcome to identify potential prognostic values of these three putative CSC markers in the same cohorts. METHODS Using immunohistochemistry, expression of nestin, CD133 and ABCG2 was analyzed in 24 rhabdomyosarcoma, 22 Ewing sarcoma and 10 osteosarcoma tissue samples and expression levels of these markers were correlated with clinical outcome. RESULTS High nestin levels indicate poor prognosis in patients with Ewing sarcoma (P = 0.001), and high CD133 expression is associated with shorter survival in rhabdomyosarcoma patients (P = 0.002). In contrast, no significant relationship was found between ABCG2 expression and the clinical outcome. CONCLUSIONS Our analysis represents the first complex study of these three putative CSCs markers together in three different types of pediatric sarcomas and showed their possible prognostic values in these tumors.

Advantages in Prognosis of Adult Patients with Ewing Sarcoma: 11-years Experiences and Current Treatment Management

Ewing sarcoma (ES) is an exceptionally rare tumor in adults. Data regarding outcomes of adult patients with ES and experiences with age-adapted therapeutic strategies are very limited. The aim of this study was to evaluate prognostic factors and clinical outcome in a cohort of adult patients treated according to pediatric protocols in the Czech Republic. The records of 58 adult ES patients diagnosed between 2002 and 2013 were reviewed and factors relevant to prognosis and survival were analyzed. The median age of study cohort was 29 years (range, 18–59). The most frequent location was axial (36.2%), followed by involvement of extraskeletal tissues (34.5%) and bones of the extremities (29.3%). Twenty-eight (48.3%) patients had metastatic disease. In cases with localized ES, the 5-year overall survival (OS) was 76.5%. Using the log-rank test, the presence of metastasis at diagnosis, local treatment without surgery and a failure to achieve complete remission were associated with significantly shorter survival. In a multivariate Cox proportional hazard analysis, the achievement of complete remission was an independent predictor of patients’s survival time. Outcomes of adults with localized ES treated according to multimodal pediatric protocols are similar to children. The achievement of complete remission is an independent predictor of survival time in ES patients. Severe hematological toxicity is foreseeable and manageable. Prognosis of patients with metastases or progression remains dismal.

Clinicopathological correlation of tumor-associated macrophages in Ewing sarcoma

AIMS Tumor-associated macrophages (TAMs) are known markers playing complex roles in tumorigenesis. However, the function of TAMs in a variety of malignancies is not yet fully understood. The aim of this pilot study was to quantify the density of TAMs in Ewing sarcoma and to determine the correlation between TAMs and clinicopathological parameters. METHODS Using immunohistochemistry, the expressions of CD68 and CD163 were examined in 24 tissue samples of Ewing sarcoma of bone. The density of CD68 and CD163-positive TAMs was analyzed quantitatively and semi-quantitatively and statistically correlated with clinical parameters. RESULTS CD163-positive TAMs outnumbered CD68-positive cells (median of 130 vs 96, respectively). No statistically significant relatio nship was found between density of CD68-positive cells, clinical parameters or prognosis. However, high levels of CD163-positive TAMs were associated with localized disease (P=0.008). In cases with a higher density of CD163-positive cells, a trend toward longer survival was revealed (P=0.063). CONCLUSION This is the first study that has quantified CD163 expression in TAMs in Ewing sarcoma and showed its possible prognostic value. CD163 was confirmed to be a more specific marker of macrophages than CD68. CD163 is not an exclusive hallmark of M2 macrophages.

Systemic AL amyloidosis with unusual cutaneous presentation unmasked by carotenoderma

Abstract We present a case study of an elderly woman with systemic lambda-type AL amyloidosis that featured unusually extensive cutaneous involvement. The case initially presented with a sudden hyper β-carotenemia with carotenoderma that instigated the clinical examination including skin biopsy. A diagnosis of systemic amyloidosis was made. Immunohistochemistry and Western-blot analysis indicated the presence of lambda light chain proteins in skin amyloid deposits. However, notable co-deposition of wild-type apoA-I and transthyretin was observed which caused initial diagnostic confusion. Proteomic analysis of microdissected skin amyloid deposits by mass spectrometry confirmed lambda light chain proteins in amyloid deposits and co-deposition of apolipoprotein A-IV and serum amyloid P-component. The patient died from renal failure caused by amyloid nephropathy combined with analgesic nephropathy. The autopsy disclosed vascular, cardiac, renal and pulmonary amyloid deposition. While all amyloid deposits were positive for lambda light chain proteins, the immunodetection of apoA-I and transthyretin varied significantly among the visceral amyloid deposits. Although the patient exhibited a 1000-fold increase in serum β-carotene levels, only a mild increase in retinol and lutein concentrations was observed. Increased β-carotene values were also found in the liver and the skin. The mechanisms underlying this hyper β-carotenemia remain undetermined.