Neuroblastoma (NB) is an embryonic neoplasm originating from the neural crest (NC), with cellular heterogeneity as one of its oncobiological characteristics (Tong et al., 2008). Recent studies have indicated that cancer stem cells in tissues and cell lines of NB can be renewed by themselves or can be differentiated multidirectionally, and they were closely related to the high malignancy of NB (Hirschmann-Jax et al., 2004). Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation. If NB is a stem cell cancer, the neuroblasts and stromal cells present in human NB tumors may represent divergent populations derived from these tumor stem cells and the relative abundance of the stem cell variants may influence their clinical behavior (Mora et al., 2001). Normal stem or progenitor cells are known to reside in most normal organs, giving rise to the differentiated bulk cells of the organ. In an analogous way, cancer stem cells are postulated to be the seeds that generate the tumor bulk, most of which is composed of primitive cells.
CD133, as a kind of transmembrane protein with a molecular weight of 120 kD, has been used as a cell marker to isolate the stem cells of NC and tumors (Shmelkov et al., 2005). A second protein present in embryonic neuroectodermal cells is the intermediate filament Nestin. Its function is to organize the cytoskeleton, but it has also been implicated in cell signaling, organogenesis, and cell metabolism (Spencer et al., 2000).
Nestin as a marker of multipotent neuroectodermal precursor cells is expressed in a cell-cycle-dependent manner and is downregulated as neuroepithelial stem cells cease division and differentiate along their respective neural or glial lineages (Thomas et al., 2004). In the adult central nervous systems, Nestin is a marker for neural stem cells lining the ventricular wall and the central canal; (Johansson et al., 1999) Nestin is also expressed by endothelial and periendothelial cells (Alliot et al., 1999). Similar to MYCN, tumor aggressiveness has been associated with elevated Nestin levels in some tumors. For instance, in primitive neuroectodermal tumors, elevated Nestin characterizes the most malignant cell type (Weggen et al., 1997). Also, Nestin protein is elevated in the infiltrating parts of highly metastatic human glioblastomas and astrocytomas (Rutka et al., 1999), and has been proposed to play a role in tumor invasion in melanomas (Florenes et al., 1994).
In the present work, the expression of these two markers, CD133 and Nestin, was studied in thirty cases of peripheral neuroblastic tumors immunohistochemically; also, their expression was correlated with other prognostic factors such as age, tumor primary site, International neuroblastoma staging system, histopathology, and MYCN status.
Materials and methods
The present study included 30 retrospective cases of peripheral neuroblastic tumors. These cases were diagnosed and treated in the University Hospital, Pediatric Oncology sector, in the period between January 2007 and December 2008. The ages of the patients ranged from 1 to 72 months.
A total of 15 cases were suprarenal tumors, nine cases were retroperitoneal tumors, five thoracic tumors, and one cervical. All the cases were formalin fixed and paraffin embedded. Hematoxylin and eosin-stained slides, pathological reports, and other medical records were reviewed to confirm the diagnosis, and clinicopathological parameters including age, sex, tumor location, tumor stage, MYCN amplification, and ploidy were recorded.
A 4-μm-thick sections were cut using a Leica microtome. Sections were transferred to adhesive-coated slides. One section was routinely deparaffinized with standard xylene and hydrated through graded ethanol in water, stained with hematoxylin and eosin, and covered with a coverslip.
For immunohistochemical staining, the positively charged slides were dewaxed by heating at 55°C for 30 min and by three 5-min washes in xylene.
Tissues were rehydrated by a series of 5-min washes in 100, 90, and 70% ethanol and PBS.
Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 20 min.
Antigen retrieval was performed by microwaving the samples for 30 min at 95°C in 250 ml 10 mmol/l sodium citrate (pH 6) for the CD133 and for 15 min for the Nestin.
The primary polyclonal rabbit anti-CD133antibody (Santa Cruz Biotechnology, Santa Cruz,California, USA) was diluted 1/100 using PBS whose pH was fixed at 6, and incubated at 4°C overnight.
Incubation was carried out at 4°C overnight with monoclonal mouse anti-human Nestin antibody (Santa Cruz Biotechnology) diluted at 1/500 in its own diluent.
After rinsing with PBS, slides were incubated at for 15 min at room temperature with biotin-conjugated secondary antibodies, followed by incubation with streptavidin-conjugated peroxidase working solution for 10 min; subsequently, the sections were stained for 10 min with 3, 3′ diaminobenzidine tetrahydrochloride, counterstained with Mayer’s hematoxylin, dehydrated, and sealed with coverslips. Negative controls were performed by excluding CD133 and Nestin antibodies during the primary antibody incubation.
CD133 and Nestin expression was evaluated semiquantitatively depending on the intensity and percentage of positively stained cells. Cytoplasmic positivity was regarded as positive staining.
Data were analyzed using the Statistical Package for Social Sciences (SPSS ver.17 Chicago, Illinois, USA).
Quantitative data were expressed using median range.
Qualitative data were described using number and percentage.
Correlation between the two quantitative variables was assessed using the Spearman rank correlation test.
The association between two qualitative variables was assessed using the Monte carlo significance test.
In all statistical tests, a level of significance of 0.05 was used, below which the results were considered to be statistically significant.
Thirty cases of peripheral neuroblastic tumors were studied. The age of the patients ranged between 1 and 72 months. A total of 15 cases were suprarenal, nine cases were retroperitoneal, five cases were mediastinal, and one case was cervical.
According to the International neuroblastoma classification 2003, they were divided into 18 cases with favorable histology and 12 cases with unfavorable histology (Figs 1–3).
According to the International neuroblastoma staging system, nine cases were in stage 1, six cases were stage 2, eight cases were stage 3, five cases were stage 4, and two cases were stage 4s.
MYCN oncogene amplification was carried out earlier and there were 11 amplified cases and19 cases without amplification.
The DNA ploidy was analyzed: 13 cases were hyperdiploid and 17 cases were diploid.
Follow-up of mortality was carried out for the patients for a period from 24 to 48 months and toward the end of the follow-up period,19 patients were living and11 patients were dead.
CD133 and Nestin cytoplasmic positivity was graded according to the number of stained cells x the staining intensity and was classified into 3 grades: 2–10% +, 11–50% ++, and 51–100% +++, and less than 2% was considered negative.
A total of 12 cases were CD +, six cases were ++, and 12 cases were +++.
A total of 10 cases were +, five cases were ++, and 15 cases were +++.
Nestin positivity was found to be highly positive in tumors of infants.
Also, Nestin was noted in ganglion cells.
There was no significant relationship between CD133 positivity and the age of the patients and the site of the primary tumor.
There was no significant relationship between Nestin positivity and age of the patients, the primary tumor site, histology, and ploidy (Figs 4–12).
Cancer stem cells have recently been proposed to be the cancer-initiating cells that are responsible for tumorigenesis and for contributing to cancer resistance in leukemia (Jordan et al., 2006). Compared with leukemia, evidence for the existence of cancer stem cells in solid tumors has been more difficult to obtain for several reasons. Cells within solid tumors are less accessible and functional assays suitable for detecting and quantifying normal stem cells from many organs have not yet been developed, and the cell surface markers required to isolate such cells have not been identified fully (Choi et al., 2009).
Recently, Hirschmann-Jax et al. (2004) used staining and flow cytometry screening to identify the cancer stem cells obtained from 23 patients with relapsed undifferentiated NB. They found that 0.8–51% of collateral stem cells existed in 15 samples that expressed several stem cell surface antigens. This finding confirmed that cancer stem cells existed in NB.
CD133, a kind of transmembrane protein with a molecular weight of 120 kD, is considered to be a hematopoietic stem cell marker mainly expressed on the CD34+ subpopulation in hematopoietic stem tissues such as the bone marrow, fetal liver, cord blood, and peripheral blood (Bhatia, 2001).
Uchida et al. (2000) separated CD133+/CD34−/CD45− immunophenotype cells from fetal brain directly and confirmed that these cells had the basic traits of nerve stem cells, self-replication, and the potential for multidirectional differentiation into neurons or gliocytes.
Singh et al. (2003) verified with immunohistochemistry and flow cytometry that CD133 was expressed in malignant medulloblastoma. They also confirmed that brain cancer stem cells could be isolated from a cell suspension by CD133 antibody immunomagnetic beads or immunofluorescence cell separation. Recent studies demonstrated the existence of CD133-positive cancer stem cells within glioblastoma that displayed a strong tumor resistance to chemotherapy. These results indicated that CD133 could be used as a stem cell marker of tumors originating from the NC (Liu et al., 2006).
The present research focused primarily on the analysis of CD133 and Nestin in peripheral NB tissue sections using immunohistochemistry. CD133, which is one of the most important cancer stem cell markers, was found to be positive in all neuroblastic tumors examined, although the frequency varied in individual samples. The high positivity was significantly correlated with unfavorable histology, advanced tumor stage, MYCN amplification, and reduced survival.
Tong et al. (2008) reported CD133 expression in NB and that the positive rate increased with the progress of the international neuroblastoma staging system degradation of differentiation degrees.
NB can be divided into three types according to cell morphology: type N (neuroblast type), type S (smooth adherence type), and type I, whose morphologic and growth traits are intercalated between these two types. Walton et al. (2004) compared 17 NB cell lines of different types and found that only type I cells expressed CD133, whereas type N and type S cells expressed slightly or did not express it.
Nestin, an intermediate filament protein, is expressed in primitive neuroepithelial cells in all regions of the central and peripheral nervous systems. Nestin was found to be expressed in all peripheral neuroblastic tumors examined, but was predominant in advanced-stage tumors and MYCN amplified tumors; also, Nestin positivity was usually noted in tumors in infants less than 1 year of age as tumors are more primitive and also some of the ganglion cells in ganglioneuroblastoma and ganglioneuroma. Many studies reported high levels of Nestin expression in malignant primary central nervous systems tumors including peripheral neuroectodermal tumors, anaplastic astrocytoma, and ependymomas, where Nestin was located in the cytoplasm tumor cells and in endothelial cells of the tumor bed (Almqvist et al., 2002).
The expression of CD133 and Nestin is significant not only for evaluating the prognosis of NB patients but also for isolating NB stem cell subgroups and understanding the mechanisms of NB development.
Conflicts of interest
There are no conflicts of interest.
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