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Transl Oncol. 2009 August 18; 2(3): 128–137.
PMCID: PMC2730138

Bcl6 Is Expressed in Neuroblastoma: Tumor Cell Type-Specific Expression Predicts Outcome1

Abstract

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and childhood. Whereas most low-risk patients do well, children with high-risk tumors often fail intensive treatment. Identification of novel biomarkers is critical to improve prognostication, tailor therapy, and develop new therapeutic targets. Differential RNA-level expression between tumor cells with neuroblastic (N-type) and Schwannian stromal (S-type) phenotypes was used to identify genes of potential interest based on tumor cell type-specific regulation. Gene expression microarray analysis revealed marked differences between N-type and S-type cells in their levels of BCL6 messenger RNA, a transcriptional regulator overexpressed in a variety of hematopoietic malignancies. S-type cells express higher levels of Bcl6 RNA and protein than N-type, and protein levels are significantly limited by proteasome function. An NB tumor tissue microarray linked to clinicopathologic data was immunohistochemically stained to measure Bcl6 protein levels. Bcl6 was detected in both the neuroblastic and Schwannian stromal regions, as distinguished histologically, and correlated with outcome. We found that expression in neuroblastic regions differentiates outcomes, in that Bcl6 expression in neuroblastic regions is associated with increased time to relapse and increased overall survival compared with absent expression in neuroblastic regions, regardless of Schwannian stromal expression. Thus, our findings suggest that Bcl6 may be useful as a prognostic marker and might represent a potential therapeutic target for high-risk NB.

Introduction

Neuroblastoma (NB), a tumor derived from neural crest cells [1], is the third most common pediatric cancer [2], accounting for approximately 7% to 10% of all childhood malignancies [3]. It is also the most common solid extracranial neoplasm of infancy and childhood [4] and accounts for approximately 15% of all childhood cancer deaths [5]. Because the clinical behavior of this disease varies widely, from spontaneous regression in very young children to the emergence of chemotherapy-resistant aggressive disease in older children with high-stage disease [6], optimal selection of treatment relies on tumor-specific characteristics that predict outcome tomaximize clinical responses while minimizing exposure to toxic drug regimens.

Contemporary practice uses risk-based treatment planning to assign patients to low-risk, intermediate-risk, or high-risk groups based on factors including age at diagnosis, tumor stage, International Neuroblastoma Pathology Committee's (revised Shimada) histopathologic classification, DNA index, amplification of the MYCN oncogene within tumor tissue, and unbalanced loss of heterozygosity for chromosome 1p or 11q [7]. Whereas patients with low-risk disease are treated with surgery alone and have a 5-year survival exceeding 95% [6], patients in the high-risk subgroup have less than a 40% chance of long-term survival despite a dose-intensive multimodal therapy [8]. The outcome for intermediate-risk patients is less certain with reports showing that subgroups can be described to have excellent survival rates when surgery is combined with nonaggressive chemotherapy, whereas others treated in the same way do poorly [9].

The heterogeneity of cell types comprising NB tumors allows for the possibility that therapeutic response is affected by differential sensitivity of tumor cell types to drugs used to treat this disease. Neuroblastic and Schwannian stromal cell-rich regions in tumors can be distinguished by histological criteria [10] and by cell type-specific marker expression [11]. For example, cells in neuroblastic regions express high levels of neurotransmitter biosynthetic enzymes [12] and the oncogenes BCL2 and MYCN. Schwannian stromal cells, however, are collagen-synthesizing cells that have little or no neurotransmitter synthetic activity [13,14]. These cell types have different and perhaps opposing roles in tumor growth. Evidence supports a range of functions for the bland Schwannian stromal cells from the production of growth-inhibitory factors that limit tumor spread to transdifferentiation into malignant-acting neuroblasts [15–18]. Most importantly, cell lines derived from high-risk NB tumors can mimic one or both of these two phenotypes that respond differently to chemotherapy in vitro [19].

The fact that NB tumors are composed of a mixed population of cells that can be distinguished both molecularly and functionally suggests that differences in gene expression between these cell types could be used to identify candidate biomarkers and therapeutic targets with particular relevance to one or the other cell type. Toward this goal, we compared the gene expression in N- and S-type NB cell lines. Among the differentially expressed genes were several genes encoding proteins already established as markers specific for either N- or S-type cells. Unexpectedly, we found that Bcl6 was among those genes with a pattern of differential expression matching known S-type cell markers. Literature review showed that Bcl6 messenger RNA (mRNA) was overexpressed in other sets of published NB microarray data [20–22], yet this protein product has not previously been reported to be expressed in NB or in other neuronal tumors. Therefore, we further studied this phenomenon and discovered that tumors with Bcl6 expression in neuroblastic cell-rich regions present more favorable outcomes. These results suggest that the expression of Bcl6 in NB tumors provides prognostic information, an association that can be further explored for use as a marker by which to stratify treatment decisions. Moreover, this work suggests the possibility that Bcl6 plays previously unexpected roles in tumorigenesis and/or treatment responses of NB.

Materials and Methods

Cell Lines

Nine NB cell lines were used for microarray analyses: five N-type (neuroblastic phenotype: IMR-32, LA1-55n, SHSY-5Y, SK-N-BE(2)-M17, and SH-IN [FSK-treated]), two S-type (Schwannian stromal phenotype: SH-EP and SMS-KCNs), and two I-type (intermediate phenotypes, with features of both N- and S-type cells: SH-IN and SK-N-BE(2)c-T38) cell lines. Twelve cell lines (SHSY-5Y, IMR-32, GOTO, SK-N-BE(2)-M17, LA1-55n, SMS-KCN-69n, SH-EP, LA1-5s, WSN, CA-2E, SK-N-SH310, and SK-N-AS) were used in Western blot analysis. Cell lines were grown in minimal essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 incubator at 37°C. IMR-32 medium was further supplemented with 1 mM pyruvate and 0.075% NaHCO3.

RNA Extraction and cDNA Synthesis

The isolation of RNA from NB cell lines was carried out using the Qiagen RNAeasy kit (Qiagen, Valencia, CA) according to the manufacturer's recommended protocols. The isolated RNA was purified with DNase (RNase-Free DNase Set, Qiagen) to digest genomic DNA according to the manufacturer's instructions. The integrity of RNA was visually assessed by conventional agarose gel electrophoresis. mRNA was reverse-transcribed into cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, Branchburg, NJ) according to the manufacturer's protocol. Parallel control reactions without the addition of reverse transcriptase were performed for each sample. The gene-specific reverse transcription-polymerase chain reaction (RTPCR) products were visualized with ethidium bromide staining after agarose gel electrophoresis.

Real-time Quantitative RT-PCR

Quantitative RT-PCR (QRT-PCR) analysis was performed using an ABI 7700 sequence detector (Applied Biosystems). For the amplification of Bcl6 and the internal control GAPDH, we used TaqMan probe labeled with 6-carboxyfluorescein from Applied Biosystems. The standard curves for Bcl6 and GAPDH were prepared using serial dilution of known concentrations of RNA (0.5,2.5,12.5, 62.5, 312.5, and 1562.5 pg) from Ramos cells (a Bcl6-overexpressing human Burkitt's lymphoma line), which was also reverse-transcribed and amplified in a similar manner as that of RNA from NB cell lines (previously mentioned). The quantity of Bcl6 mRNA was calculated after normalizing for the internal control GAPDH.

Immunoblot Analysis

Nuclear protein (20 µg) from NB cell lines was resolved by 8% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Invitrogen, Grand Island, NY). Membranes were blocked with 5% milk and immunoblotted with anti-Bcl6 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) at a 1:1000 dilution. The blots were visualized by chemiluminescence using a horseradish peroxidase-linked anti-rabbit immunoglobulin G secondary antibody (GE Healthcare, Buchinghamshire, United Kingdom) diluted at 1:5000. Anti-cAMP response element-binding protein (Upstate Biotechnology, Lake Placid, NY) was used as a loading control. For experiments evaluating Bcl6 posttranslational processing, cells were pretreated for 18 hours with specific proteosome inhibitors MG132 (10 mM; Calbiochem, La Jolla, CA) dissolved in dimethylsulfoxide or bortezomib (50 nM; Velcade, Millenium, Boston, MA).

Oligonucleotide Microarray Hybridization and Analysis

Preparation of cRNA from total RNA, hybridization, scanning, and image analysis were performed according to the manufacturer's protocol and as previously described [23]. We used commercially available oligonucleotide arrays (HG_U133A; Affymetrix, Santa Clara, CA) containing 22,283 probe sets. Each probe set typically contains 11 perfectly matched 25-base-long probes (PMs) as well as 11 mismatch probes (MMs) that differ from the PM probes at the central base. Using publicly available software [24], we computed the average of the PM - MM differences for each probe set on each array after discarding the largest and smallest 20% of the PM - MM differences, and scaled the data for each array to give a mean of 1500 units. Data were then quantile-normalized to make the values of 99 evenly spaced quantiles (at 0.01, 0.02, …, 0.99) agree across all of the arrays.We log-transformed values using log [max(x + 50; 0) + 50]. Conservative average fold differences were computed based on the differences in log-transformed data.

Tissue Microarray Construction

Tissue specimens were obtained from the University of Michigan Department of Pathology archives under the approval of the Institutional Review Board. Tissue arrays were constructed using three 1.0-mm cores taken from 48 paraffin-embedded, formalin-fixed neuroblastic tumors (32 NBs, 10 intermixed and nodular ganglioneuroblastomas (GNBs), and 6 ganglioneuromas (GNs)), 5 normal adrenal glands, and 7 other neural crest-derived (melanoma, pheochromocytoma, paraganglioma, and schwannoma). The histologic classification of archived tumor specimens was available, but all specimens were blindly reviewed by a single pathologist (J.A.J.) and classified according to current standards using the International Neuroblastoma Pathology Committee's classification [10,25]. Core sampling was performed to ensure that optimal representation of both neuroblastic and stromal components was present. Completed array blocks were cured at 37°C for at least 24 hours, then heated at 52°C for 15 minutes, and rapidly cooled in an ice water bath. Multiple 5-µm tissue sections were cut from each block, with every 10th section stained with hematoxylin/eosin formorphologic assessment and comparison to adjacent immunostained levels.

Protein Expression Analysis

Immunohistochemistry was performed using commercially available monoclonal antibodies and standard manufacturer's protocols on a Ventana automated stainer. Briefly, slides were incubated with anti-Bcl6 (PG-B6p; Dako Cytomation, Carpinteria, CA) at a 1:20 dilution after a 20-minute pretreatment in a high-pH buffer at 95°C. Signals were evaluated for tumoral distribution (neuroblastic cells vs Schwannian stromal cells), cellular distribution (nuclear vs cytoplasmic), and intensity (semiquantitatively graded as 0, 1+, 2+, or 3+). The neuroblastic component was defined as any or all of the following: neuroblasts, immature gangliocytoid cells, and mature ganglion cells; the stromal component was defined as spindled cells with scant clear to eosinophilic cytoplasm embedded in a loose connective tissue matrix resembling Schwann cells or perineurium. Specimens excluded from analysis included those tumor array cores with insufficient tissue or missing sections, those that were necrotic, and those with insufficient neuroblastic or stromal components for evaluation. For each evaluated specimen, the mean neuroblastic component staining, the mean stromal component staining, and the mean component staining difference were calculated for all three cores.

Patient Data

Tumor array specimens from 48 patients were evaluable and included in the analysis. Patient characteristics included tumor specimens from 23 females and 25 males, ranging in age from 0 to 16 years. The mean age at diagnosis was 3.0 years, and the median age was 2.5 years.Twenty-five tumor specimens were from stage I or II patients, 5 from stage III patients, and 17 from stage IV patients. There was one stage IVS patient included in this analysis. Length of available follow-up data ranged from 1 to 17 years from the time of diagnosis. Thirty-seven patients were alive at the time of the study with mean follow-up of 6.7 years. Of the 32 NB patients, 13 had favorable histologic diagnosis and 19 had unfavorable histologic diagnosis. Of the 10 GNB patients, 7 had favorable histologic diagnosis, 2 had unfavorable histologic diagnosis, and 1 patient had missing data precluding proper classification. The GN group included six patients, all with favorable histologic diagnosis.

Statistical Analysis

The goal of the analysis was exploratory in nature. Clinical outcomes were correlated to the intensity of the expression and the presence of the expression in neuroblastic and stromal regions. For Bcl6 intensity analysis, the subject-level average was used in correlation with the outcomes. Subject-level average was computed by averaging the intensity of Bcl6 expression from cores on tissue microarray that were from the same subject. The intensity in the neuroblastic and stromal regions and the differential expression between neuroblastic and stromal regions were evaluated. For the analysis on the presence, Bcl6 expression was grouped into four categories based on the presence (+) or absence (-) of expression in S-type cells and N-type cells. The four possible groupings were as follows: S+/N+ (both stromal and neuroblastic cell Bcl6 expression), S+/N- (stromal cell expression only), S-/N+ (neuroblastic expression only), or S-/N- (both stromal and neuroblastic cells negative). Covariates of interest included: MYCN (amplified, indeterminate, and normal), clinical stage (I, II, III, and IV), and tumor histologic diagnosis (NB, GNB, or GN). Outcomes analysis included Bcl6 expression in relation to overall survival and time to recurrence from end of therapy (event-free survival). Spearman rank correlation coefficient was used to assess univariate association between Bcl6 expression intensity and covariates of interest, whereas Fisher exact test was used to assess univariate associations between four-group N and S regions presence of Bcl6 expression and covariates of interest. The Kaplan-Meier method and log-rank test were used to compare the homogeneity of survival rates between categories of Bcl6 expression, the intensity of Bcl6 expression, as well as discrete covariates. Time to recurrence was defined as the time when NB relapse was documented. Subjects who did not recur at last follow-up or who died for reasons other than NB recurrence were censored from the analysis. All statistical analyses were done using SAS v9.0 (Carey, North Carolina). A 2-tailed P value of 0.05 or less was considered to be statistically significant.

Results

Bcl6 Is Expressed in NB

RNA expression profiles of nine NB cell lines (five N-type, two S-type, and two I-types) and three NB tumors were generated using the Affymetrix platform. Genes that were differentially expressed between N- and S-type cell line groups with a fold change ≥5 (P < .01) were identified yielding 411 probe sets that included several genes already known to be expressed specifically in either the neuroblastic or the stromal components of neuroblastic tumors, validating the strategy of using differential expression to identify cell type-specific gene expression. Results in Figure 1 show expression data from a select subset of these genes. As expected, vimentin, collagen type IV, and fibronectin, known stromal markers [26], were expressed at significantly higher levels in S-type cell lines, whereas neurofilament and chromogranin A, markers of neuroblastic cells [27], were expressed at higher levels in N-type cells. Surprisingly, the expression of Bcl6, a transcriptional regulator implicated in B-cell development and lymphomagenesis [28], was differentially expressed with a high-level expression in S-type cells, having an N/S ratio of 0.05, similar to known S-type-specific markers. Moreover, Bcl6 expression was detected in each of the three NB tumor specimens (NB1, NB2, and NB3 in Figure 1), which contained mixed populations of stromal and neuroblastic cells.

Figure 1
Differential expression of N- and S-type marker genes and Bcl6. Expression level of Bcl6 and selected RNA previously shown specific for neuroblastic (top panel) or Schwannian stromal cells (bottom panel) in a panel of N-type (IMR-32, LA1-55n, SHSY-5Y, ...

Bcl6 mRNA Expression in an Expanded Panel of NB Cell Lines

To confirm the presence and differential expression of Bcl6 mRNA in NB cells, we isolated RNA from a larger panel of 12 well-characterized NB cell lines (6 S-type and 6N-type). RNA was reverse-transcribed, and cDNA was amplified using either Bcl6 or β-actin-specific primer sets as described in the Materials and Methods section. Consistent with our microarray results, ethidium bromide-based detection of PCR products identified Bcl6 message in each of the NB cell lines analyzed (Figure 2A). As a group, expression in S-type cell lines seemed higher than in N-type cell lines, although Bcl6 expression was not exclusive to S-type cells. In several S-type lines, the level of Bcl6 expression was similar to the level in Ramos cells, a human Burkitt's lymphoma cell line in which Bcl6 expression controls growth and differentiation [29,30].

Figure 2
Bcl6 RNA expression in N- and S-type NB cell lines. (A) Reverse transcription-polymerase chain reaction products of Bcl6 RNA in NB cell lines detected by ethidum bromide staining. Lanes 1 to 6 and 7 to 12 are from the indicated N- and S-type cell lines, ...

These results were confirmed by using QRT-PCR with normalization to GAPDH as the internal control. As shown in Figure 2B, there was a significantly higher expression of Bcl6 mRNA in S-type cells (filled bars) than N-type cells (striped bars; P < .008). The difference observed between N- and S-type cells for Bcl6 is similar in magnitude to that of vimentin (Figure 2C), an S-type-specific marker [26].

Bcl6 Protein Expression in NB Cell Lines

Initial attempts to detect Bcl6 protein in NB cell lines showed that even in cell lines with high levels of Bcl6 mRNA, Bcl6 protein levels were barely detectable using standard immunoblot analysis techniques. Previous reports suggest that Bcl6 protein expression in B cells is tightly regulated by proteosomal processing [31]. Therefore, we hypothesized that proteosomal degradation may limit Bcl6 protein levels in NB cell lines. To test this possibility we treated cells with the proteosomal inhibitors MG132 and bortezomib, both reversible inhibitors of the chymotrypsin-like activity of the 26S proteasome [32,33]. Cells were pretreated for 18 hours with the inhibitors before extracts were made, and Bcl6 expression was determined. As shown in Figure 3A, Bcl6 protein levels increase in response to proteasome inhibition to an extent that varies according to cell line and the inhibitor used. For example, in SH-EP and SK-N-SH cells, Bcl6 expression increased in response to MG132 but not bortezomib treatment. Interestingly, pretreatment of N-type cell lines with proteasome inhibitors did not affect Bcl6 protein levels. These results suggest posttranslational processing leading to proteosomal degradation may contribute to the regulation of Bcl6 in NB similar to B-cell lymphoma [34].

Figure 3
Inhibitors of the proteasome increase Bcl6 protein in NB cells. Immunoblots of nuclear extracts to detect Bcl6 protein in S-type cell lines (A) and N-type cell lines (B) either untreated or treated as indicated with MG132 (10 mM) or bortezomib (50 nM) ...

Bcl6 Protein Expression in NB Tumors

Next, we extended our analysis to determine whether Bcl6 protein was expressed in NB tumor tissue, and if so, whether there was differential expression between neuroblastic and Schwannian stromal cell-rich regions. Tumor tissue microarrays comprising a panel of primary NB tumors, normal adrenal glands, and other selected neural crest-derived neoplasms were subjected to immunohistochemistry to detect Bcl6 protein. Bcl6 staining was evaluated by scoring the signal intensity using a grading system from 0 (no staining) to 3+ (marked expression). Furthermore, in primary NB tumors, a separate score was determined for neuroblastic and Schwann/stromal cell-rich regions.

Bcl6 is detected in normal adrenal glands at an average staining intensity of 2.6, that is significantly greater than expression in either neuroblastic (average intensity = 1.35) or stromal regions (average intensity = 1.0) of NB tumors. Bcl6 expression in benign neural crest tumors (paraganglioma, pheochromocytoma, and schwannoma) is also greater (average intensity = 2.9) than in NB. Average expression in melanoma, another malignant neural crest derived tumor, is 0.7. These results suggest a pattern where malignant neural crest tumors have lower Bcl6 expression than normal adrenal or benign tumors derived from the neural crest.

Bcl6 protein expression in the NB tumor cores was correlated with clinical and pathological factors relevant in establishing risk group as well as predicting outcome. Forty-eight patients with available follow-up data were included in this analysis. Median duration of follow-up was 5.0 years (range, 1.3–17.5 years). Thirty-eight (79%) patients were alive, 33 of 38 were in their first complete remission, and 5 of 38 who had relapsed were in a subsequent complete remission. Ten patients (21%) had died: nine of NB dissemination and one of treatment complications. Of the 48 tumors included in this array, Bcl6 protein was detected in 50% (24/48).

To determine whether expression varied between neuroblastic and Schwannian stromal components, a subset of tumor samples (Table 1) was selected based on copresence of sufficient neuroblastic and Schwannian/stromal-rich regions to adequately assess expression in each component separately (n = 20). The absence of either neuroblastic or stromal regions in the remaining samples (n = 28) was a result of technical limitations whereby core sampling did not include a representative heterogeneous specimen or in two instances where the primary tumor did not contain any histologically definable stromal component. Table 1 shows a summary of Bcl6 staining according to cell type and tumor histologic diagnosis. Of the 20 patient tumors with both neuroblastic and Schwannian components, 12 were classified as NB, 5 as GNBs, and 3 as GNs. This secondary analysis was completed by tabulating whether Bcl6 was expressed in Nand S regions separately and assigning tumors to one of the following groups: N+/S+ (Bcl6 expression in both neuroblastic and stromal regions), N+/S- (only neuroblastic regions positive), N-/S+ (only stromal regions positive), or N-/S- (no Bcl6 expression in either region). Figure 4 shows a representative staining pattern for each of the four subgroups. In 18 of 20 specimens, Bcl6 protein was expressed in at least one of the histological subsets (neuroblastic or stromal). In five tumors, expression was detected in both neuroblastic and stromal regions (N+/S+). In eight tumors, Bcl6 expression was detected only in neuroblastic regions (N+/S-), and in five tumors, Bcl6 expression was confined to the stromal regions (N-/S+). The intensity of Bcl6 staining, when present, did not differ significantly between the neuroblastic (average intensity = 1.35) and stromal regions (average intensity = 1.0). Thus, although the pattern of Bcl6 expression in vitro showed increased expression in S-type compared with N-type cell lines, Bcl6 expression within tumor tissue was neither specific to nor increased in the cells comprising the Schwannian stromal cell-rich regions.

Figure 4
Bcl6 is expressed in human NB tumors. Bcl6 protein expression in NB tumor samples was detected by immunostaining. Tumors are classified as either N+/S+, N+/S-, N-/S+, or N-/S- based on the detection of Bcl6 (nuclear brown straining) in neuroblastic (green ...
Table 1
Bcl6 Expression in Neuroblastic (N) and Schwannian Stromal (S) Tumor Regions According to Histologic Diagnosis.

The intensity of Bcl6 expression in either tumor component was not significantly associated with overall survival (data not shown). We then compared Bcl6 expression with the known prognostic variables important for NB risk stratification individually (MYCN status, stage, and histologic diagnosis). There was no significant correlation between Bcl6 expression and MYCN status or stage; however, a weak but significant correlation between Bcl6 expression and histologic diagnosis was demonstrated using Spearman correlation. In this analysis, Bcl6 expression in neuroblastic regions was associated with favorable histologic diagnosis. Conversely, increased Bcl6 expression in stromal regions was associated with unfavorable histologic diagnosis. Importantly, when we compared the association between Bcl6 groups (N+/S+, N-/S+, N+/S-, and N-/S-) and patients' overall survival, Kaplan-Meier analysis demonstrated that patients whose tumors had Bcl6 expression in the neuroblastic cells (N+/S+ and N+/S-) had a more favorable outcome measured by overall survival (P = .038) (Figure 5A). This similar trend was observed in the time to recurrence outcome, but P value did not reach statistical significance at the 0.05 level (P = .065; Figure 5B).

Figure 5
Bcl6 expression in neuroblastic tumor regions is associated with better outcomes. Kaplan-Meier analysis of survival (A) and time to recurrence (B) shows that Bcl6 expression in neuroblastic regions is correlated with increased overall survival (P = .038) ...

Discussion

This work identifies the presence of Bcl6 RNA and protein in NB cells and human NB tumor tissue. To our knowledge, this is the first report to identify the presence of Bcl6 protein in NB and that its pattern of expression is associated with prognostic variables and outcomes.

Bcl6 is a transcriptional repressor that binds DNA through six zinc fingers [35,36] and regulates transcription by interacting with a number of other factors including Jun proteins, nuclear receptor corepressor, Bcl6 corepressor, silencing mediator of retinoid and thyroid receptor, and histone deacetylase family proteins through its amino-terminal BTB domain (bric-a-brac, tramtrack, broad complex) [37–40]. Bcl6 has an important role in B-cell development. Its expression is low in immature B cells, increases as these cells differentiate through the germinal centers, and is then turned off when B cells terminally differentiate into plasma cells [41,42]. In germinal centers, Bcl6 delays terminal differentiation and allows cells to survive DNA double-strand breaks that occur as the B-cell receptor gene is rearranged. Mechanistically, this is accomplished by repression of ATR and TP53 transcription that otherwise blocks B-cell proliferation and induces cell death [39,43].

By maintaining the proliferative potential of cells with DNA damage, Bcl6 can support neoplastic transformation, consistent with observations that its expression is frequently increased in hematologic malignancies. For example, Bcl6 overexpression is detected in 40% of diffuse large B-cell lymphomas (DLBCL) [44], 14% of follicular lymphomas [45], and 48% of nodular lymphocyte-predominant Hodgkin lymphomas [46]. In vivo models of lymphomagenesis have confirmed Bcl6's oncogenic potential in this cell lineage. “Knock-in” experiments in mice that cause Bcl6 overexpression in mature B cells result in lymphomas with features resembling human DLBCL [47].

As Bcl6 is primarily associated with normal and abnormal B cell development, its presence in half of the examined NB tumors was unexpected. Gene expression profiling and immunostaining studies document that Bcl6 is detected in a limited number of other types of cancer including certain skin tumors [48] and breast carcinoma, where its expression is associated with increased cell survival and arrest of differentiation [49].

Whether Bcl6 has a role in NB pathogenesis needs to be determined, although its abilities to protect cells against DNA damage-induced death and to block differentiation are of clear potential relevance to NB. Neuroblastoma develops from neural crest cells in which complete differentiation along the sympathoadrenal lineage is blocked [50]; therefore, it is tempting to speculate that analogous to its role in arresting B-cell differentiation in normal germinal centers and in DLBCL, Bcl6 arrests the differentiation of neural crest cells as a step in the development of NB tumors. Bcl6 attenuation of the DNA damage response mechanism could promote the genetic instability characteristic of NB tumors. Understanding the significance of Bcl6 expression in NB tumor cells will also be furthered by determining the pattern of Bcl6 expression during normal sympathoadrenal lineage development to ascertain whether it has a function similar to B cells in blocking terminal differentiation.

Although the Bcl6 locus (3q27) has been reported in chromosomal translocations observed in NB [51], 3q abnormalities are not common in this cancer, and the region encompassing 3q27 is amplified in only 6% of NB tumors [52]. Thus, chromosomal changes do not commonly affect Bcl6 expression in NB. Additional experiments are needed, however, to determine whether point mutations or small deletions in key regulatory sequences within the Bcl6 gene occur in NB, similar to those found in B cells that may contribute to its abnormal regulation in lymphoma [53–55].

Our results indicate that Bcl6 expression in the neuroblastic component of NB tumors is associated with low-risk disease and better clinical outcomes. Bcl6 expression in primary central nervous system lymphoma, DLBCL, and follicular lymphoma is similarly associated with better outcomes, including improved overall survival [44,56–58]. Thus, in lymphoma where Bcl6 can clearly promote tumorigenesis, its expression in established disease is, nevertheless, an indicator of favorable outcomes. One possible explanation for this relationship between expression and outcome is that Bcl6 increases responsiveness to chemotherapy. Because Bcl6 attenuates the DNA damage response that normally arrests cell cycle progression, its expression may increase drug killing by preventing the repair of DNA damage induced by chemotherapy resulting in replication failure. Work is underway to determine whether Bcl6 expression affects chemosensitivity and DNA repair in NB cell lines. Preliminary experiments using small interfering RNA to knock down Bcl6 in S-type cell lines have shown that reducing RNA level by more than 50% has no effect on survival or growth, demonstrating the feasibility of using this model to study chemoresponsiveness.

Finally, the findings presented here identify opportunities for future work to elucidate whether Bcl6 expression improves classification for the purposes of treatment stratification, to determine whether Bcl6 expression can be regulated to treat this disease and to define the role of this protein in NB tumor development.

Footnotes

1This work was supported in part by the National Institutes of Health grants DK067102 (R.P.S.K.) and CA10456 (A.W.O.), the Jeanette Ferrantino Hematology Research Fund (V.P.C.), the Ravitz Foundation (V.P.C.), and the A. Alfred Taubman Medical Research Institute (V.P.C., R.P.S.K., and A.W.O.). This work used the sequencing core of the Michigan Diabetes Research and Training Center, which is funded by the National Institute of Diabetes and Digestive and Kidney Disease NIH5P60 DK 20572.

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