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Neuroblastic and Schwannian Stromal Cells of Neuroblastoma Are Derived from a Tumoral Progenitor Cell¹

Departments of Pediatrics [J. M., N-K. V. C., I. C., S. C.] and Pathology [J. M., G. J., P. I., M. A., M. L., C. C-C., W. L. G.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NB³ is a pediatric cancer that arises from precursor cells of the peripheral sympathetic nervous system. Biologically, it has a wide range of behavior, including spontaneous regression/involution, spontaneous or induced maturation/differentiation, and aggressive proliferation. Spontaneous or induced differentiation of NB could recapitulate neural crest development with the formation of cells along multiple lineages (1 , 2) . In fact, histologically mature NB consists of two main cell populations, neuroblastic/ganglionic cells and non-neuronal stromal cells, including Schwann, perineurial, and satellite cells.

It is well established that the presence of SS in NB is age- and stage-related. Typically, infants with stage 4 tumors (International Neuroblastoma Staging system; Ref. 3 ), as well as aggressively proliferating tumors (International Neuroblastoma Staging system stage 4), are histologically SS poor (4 , 5) . Abundant SS is frequently seen in LR NB tumors with maturation potential and correlates closely with favorable genetic findings, i.e., triploidy (6 , 7) . The origin of the stromal component and whether the presence of stroma is a cause or a consequence of the maturation potential of tumor cells is not yet fully established.

The coexistence of neuroblastic and SS cells in maturing NB and the appearance of Schwannian-appearing cells in NB cell cultures led to the hypothesis that NBs originate from pluripotent neural-crest cells. In vitro, three related cell types exist: the S-type with characteristics of immature Schwannian, glial, or melanocytic cells; the N-type with neuronal characteristics, and the I-type neural crest stem cells (8 , 9) . An alternative hypothesis was suggested by Ambros et al. (10) in 1996. They reported that SS cells in maturing NB lacked the genetic anomalies found in the neuroblastic cells and hypothesized that the SS cells in NB were reactive in nature and have been recruited from the surrounding nonneoplastic tissues. On the basis of known interactions between neuroblasts and their supporting stroma, they proposed that the recruited normal SS cells may be producing antiproliferative- and differentiation-inducing factors leading to maturation of neoplastic cells.

However, other studies of advanced stage disease suggest otherwise, e.g., stromal-like cells derived from bone marrow metastases after temporary passages in vitro share some of the genetic abnormalities with the neuroblastic counterpart, suggesting a common tumoral origin (11) . In addition, genetic analysis of homogeneous populations of cells isolated by LCM from diploid stage 4 NB showed identical genetic alterations in both SS and neuroblastic components (12) .

In this study, we analyzed the clonal origin of the neuroblastic and non-neuronal cell subtypes (identified by morphology as SS) from fresh surgical specimens. To overcome the complexity and heterogeneity of three-dimensional structure of tissues, three different approaches were taken: (a) LCM was used to obtain histologically homogeneous cell subtype populations for analysis of 1p36, 11q, 14q, and 17q allelotype and MYCN copy number; (b) LSC was used to provide multiparametric information about morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor FISH in cases where touch imprints of the two cell types could be manually microdissected.

	MATERIALS AND METHODS

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Tumor Tissues and Clinical Data.
Forty frozen tumor specimens each with >75% tumor cell content, obtained from 17 patients with stage 4 NB patients and 23 patients with LR NB, were studied. Tumors (28) were used for LCM analysis, 20 for the LSC experiments, and 7 for FISH analysis. Human tissues were used for biological studies, according to the guidelines of the institutional review board. Clinical and biological characteristics for all cases are summarized in Table 1 .

Allelic analysis of samples was performed using polymorphic microsatellite markers D1S548, D1S1592, D1S2826, D1S552, and D1S511 for chromosome arm 1p36; D11S1982, D11S488, D11S1294, D11S1302, and D11S1366 for chromosome arm 11q23; and D14S544, D14S120, D14S302, D14S140, D14S119, and D14S129 for chromosome arm 14q32. To investigate chromosome gain at 17q, we followed the microsatellite-based approach of Janoueix-Lerosey et al. (15) using D17S796, D17S938, D17S1176, and D17S786 at chromosome 17p13 and D17S693, D17S916, D17S674, D17S1304, and D17S668 at chromosome 17q25. Alleles were measured using an Applied Biosystems model 310 automated fluorescent DNA sequencer (Applied Biosystems, Foster City, CA) with Genescan software (16) . LOH was determined by comparison of the allelic ratio between the normal and tumor or LCM specimen in heterozygous samples as described previously (17) .

Relative quantitation of MYCN and the endogenous control ß-actin was performed by real-time quantitative PCR using an Applied Biosystems Prism 7700 Sequence detection system. The primers and probe were designed using the applications-based primer design software primer express (Applied Biosystems). PCR protocol was performed according to the manufacturer guidelines. Every PCR run included a five-point standard derived from serial dilutions of NB cell line LAN-1 DNA to generate a standard curve for MYCN and ß-actin, plus a no-template control. For each unknown test sample, the amount of MYCN and endogenous ß-actin was determined from the respective standard curve. Dividing the MYCN level by the ß-actin level resulted in a normalized MYCN value.

Immunocytochemical Detection of GD2 and S-100.
Touch imprints were obtained after 10 consecutive previous touch preparations from frozen tumor blocks to eliminate previously cut cells. Immunocytochemical detection was performed following standard methods (18 , 19) . The slides were fixed in 80% ice-cold ethanol at -20°C for 24 h, washed twice with PBS containing 1% BSA and 0.1% sodium azide PBS-BSA, and blocked for 30 min with 5% normal goat serum in PBS. Then cells were incubated overnight in the dark at 4°C with 100 µl of PBS-BSA containing 2 µg/ml mouse anti-GD2 monoclonal antibody (3F8), a recognized marker for NB cells (20) , 2 µg/ml anti-S-100 monoclonal antibody (BioGenex Laboratories, San Ramon, CA), or 2 µg/ml mouse anti-IgG as a negative isotype control. Slides were then washed in PBS-BSA and incubated with 100 µl of secondary antibody conjugated to FITC (Dako, Carpinteria, CA) for 30 min in the dark at room temperature. Finally, the cells were stained in PBS containing 5 µg/ml PI and 100 µg/ml RNase A for 30 min at room temperature. The slides were then mounted under coverslips in PBS for analysis by LSC.

The specificity of the immunofluorescence staining was confirmed by a titration of the monoclonal antibodies GD2 and S-100. Normal lymph node (negative control), tumor samples, and one NB cell line (SK-N-ER; positive control) were used. Five dilutions were tested ranging from 0.2 µg/ml to 3.3 µg/ml. Two µg/ml was the selected concentration based on the lack of staining in lymph nodes and the highest fluorescence intensity in the positive control (tumor samples). Concentrations as high as 3.3 µg/ml, however, did not show increased nonspecific staining in lymph nodes or improved staining in the tumor samples.

LSC.
Cytometric measurements were performed using a laser scanning cytometer (LSC 101; Compucyte Corp., Cambridge, MA) as described previously (21) . The excitation wavelength was 488 nm. Green (FITC) and red (PI) fluorescences were measured by separate photomultipliers within 530 ± 20 nm and 600 ± 20 nm spectral ranges, respectively. An additional light source provided transmitted illumination, which was used to visualize the cells through an eyepiece or a CCD camera (21 , 22) . At least 3000 cells were analyzed per sample.

FISH.
Bicolor FISH studies were performed on touch imprints obtained from manual microdissection of stromal and neuroblastic-rich areas using probe mixtures of the directly labeled centromere-specific alphoid sequences for chromosomes 1 and 17 (CEP1 and CEP17; Vysis, Downer’s Grove, IL) and a mixture of two probes localized to sub-regions of 17q25 (525L23 and 497H17 RPC11 human bacterial artificial chromosome). One µg of each DNA probe was directly labeled using a nick translation kit (Vysis) according to the manufacturer’s instructions. The labeled DNA was then coprecipitated for annealing purposes with 2 µg of Cot-1 DNA (sonicated total human DNA). The telomeric location of the probes was confirmed by hybridizing to metaphase spreads of normal human lymphocytes in a bicolor assay with directly labeled centromere 17-specific alphoid sequence (CEP17; Vysis). FISH was performed according to standard protocols and previously published studies (23) . Images were prepared using the Applied Image Analysis System (Applied Imaging, Pittsburgh, PA). The number of hybridization signals for each probe was assessed in a minimum of 200 interphase nuclei with strong and well-delineated signals. As controls, normal peripheral blood lymphocytes were simultaneously hybridized with these probes.

	RESULTS

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Allelotype and MYCN Analysis in LCM Specimens.
Neuroblastic and SS components of the tumor were identified by morphology and isolated by LCM (Fig. 2) . LCM samples adequate for allelic analysis were obtained in 28 (12 LR NB and 16 stage 4) samples. Twenty-seven of the 28 cases (96%) showed identical genetic features in the whole tumor and individual cellular components. Table 2 summarizes the results of the allelotype analysis for all of the subpopulations obtained by LCM. Figs. 1 and 2 show examples of AI and LOH electropherograms obtained for the microdissected neuroblastic and SS specimens from tumors 2140, 1381, 1940, and 1163.

View larger version (68K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Allelotype and FISH analysis of the LR tumor 2140 show triploidy of chromosomes 1 and 17 for both cell subtypes. a, photomicrographs showing histology of neuroblastic-rich region (left) and SS-rich area (right) of tumor 2140 before LCM. b, neuroblastic cells (left) and SS cells (right) obtained after LCM and subsequently used for allelotype analysis. Center panels, the AI detected by allelotype analysis secondary to the triploidy for microsatellite marker D1S547 at chromosome 1q: N, normal (blood); T, whole tumor; Nb, LCM neuroblasts; and Str, LCM SS cells. c, FISH analysis using chromosome 1 and 17 centromeric probes (red and green, respectively) of touch imprints obtained after manual microdissection of the neuroblastic and SS-rich areas of tumor 2140 showing triploidy for both chromosomes (three green and red signals).

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative electropherograms showing examples of retention of heterozygosity (center), LOH (left panel), and AI secondary to triploidy (right panel) for the LCM neuroblastic (Nb) and Schwannian-Stroma (Str). X axis, allele bp size; Y axis, relative immunofluorescence intensity.

In 1 MYCN-amplified tumor (tumor 1381), we were able to obtain enough sample for comparison of MYCN copy number between neuroblastic and SS cell subtypes by quantitative PCR. On the basis of eight replicative quantitations, the patient’s normal tissue control MYCN copy number was 1.3 ± 1.8, whereas the LCM neuroblast sample had 360 ± 297, and the LCM SS sample had 106 ± 157 MYCN copies.

Cytometric Analysis.
LSC was used to evaluate the DNA ploidy in vivo on each cell subtype. SS cells were identified by morphology and independently by indirect immunofluorescence with the S-100 monoclonal antibody. The tumor-specific anti-GD2 monoclonal antibody 3F8 was used to mark tumor clones (20) . 3F8 binds strongly to NB, whereas it is unreactive with most normal human tissues, including blood and marrow (20 , 24) . LSC analysis was performed in 19 LR tumors because abnormalities of DNA index are common in LR NB and 1 stage 4 tumor with multiple clones identified by FISH for MYCN (Tumor #2123, Table 1 ). The results are summarized in Table 3 .

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. DNA content and cytology of GD2-positive cells in NB. Left panels, DNA content distributions of two tumor samples. The percentage of cells in each peak was estimated after staining of cellular DNA with PI and fluorescence measurement by LSC. Center panels, bivariate distributions (scatterplots) of GD2-immunofluorescence versus DNA content of the same sample. The analytical gate was set as described in "Materials and Methods." Arrows, the ploidy peaks. Tumor 2123 shows three discrete ploidy peaks, one diploid and two hyperploid, all GD2 positive. Tumor 1163 shows a small diploid and a prominent hyperploid peak, all GD2 positive. After LSC measurement, cells that highly expressed GD2 were selected for viewing. Characteristic small-round cell morphology for neuroblastic cells was confirmed, and a gallery of digital images was created (right panel).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Multiparametric analysis by LSC of S-100 expression, DNA content, and nuclear cell morphology. DNA content distribution of tumor sample 2132 shows two discrete ploidy peaks: one diploid and one hyperploid (left panel). Bivariate distribution of S-100 immunofluorescence versus DNA content of the same sample shows S-100-positive, hyperploid cells (center panel). Cells that highly expressed S-100 were gated and selected for viewing (right panel). Cells with spindle morphology (SS) were detected in the fields.

	DISCUSSION

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One major limitation of using human tumors for clonal studies is the difficulty in partitioning complex mixture of different cell types in the three-dimensional structure of tissues. To overcome these technical difficulties, we combined fluorescent cytometry and image and genetic analysis with advanced microdissection laboratory technology. LCM allows the procurement of homogeneous cell populations from heterogeneous tissue (13) . Using LCM samples, we analyzed both neuroblasts and SS cells in isolation. Our results show unequivocally that both cell components of individual tumors are genotypically identical and tumor derived. In addition, we used LSC, a microscope-based cytometer that provides the unique advantages of image analysis and cytometry measurements on intact single cells (21) . Using LSC, we could show that stroma cells identified by morphology and S-100 immunostaining showed the same GD2 immunoreactivity and DNA index as corresponding neuroblasts from the same tumor.

Because FISH can convincingly mark chromosome gains or losses in individual cells, the presence of aberrant signals is often accepted as proof of tumoral origin. Ambros et al. (10) showed that Schwannian cells from NB with maturing histological features lacked the chromosome 1p36 triploidy found in the neuroblastic cells, leading to the hypothesis that the Schwann cells were normal cells recruited into the tumor during the maturation process. However, the absence of evidence cannot be equated as the evidence of absence given the heterogeneity inherent in most human tumors. Furthermore, FISH analysis on cut paraffin sections as used by Ambros et al. are sometimes difficult to interpret because of the high degree of cellular heterogeneity and randomly sectioned nuclei, requiring statistical estimates of DNA content. This may explain the differences between our findings and previous studies (10) . In contrast, FISH carried out on touch imprints of separate neuroblastic and stromal areas of tumors avoids many of these pitfalls. Unfortunately, the intimate admixture of tumor and stroma cell subtypes in most human tumors precluded the use of this technique for most cases. In all evaluable cases, FISH analysis of touch imprints unequivocally detected identical chromosomal makeup for neuroblast and corresponding SS cells

LR NB is characterized clinically by a lack of metastatic potential, a propensity for maturation (25) , and various degrees of differentiation at the histological level. In one previous study, we found that near triploid DNA index was the best predictive marker of progression-free survival in LR NB independent of the presence or absence of significant stromal component (7) . The data provided in this study show that most cases of LR NB have a triploid DNA index in the neuroblastic as well as in the stroma compartments.

In contrast, NB tumors with aggressive proliferation and metastatic behavior (stage 4) are characterized by diploid DNA index, structural chromosomal aberrations, and MYCN gene amplification (26) . Histologically, they are mostly stroma poor (5) . After cytotoxic therapy, a significant percentage of stage 4 cases are left with or acquire a differentiated stroma. In this group of tumors, the presence of abundant stroma had no survival significance in our series (data not shown), as well as in other reports (27) . Our results show that both neuroblasts and stromal cells have the same genetic composition and suggest that current cytotoxic therapies may either induce the maturation of neuroblastic cells to undergo non-neuronal differentiation or fail to kill the stromal elements. In this context, the in vitro observation that S-type cells can trans-differentiate into N-type cells under certain pressures takes particular significance (28) . If this phenomenon occurs in vivo, the late relapses commonly seen in stage 4 cases postchemotherapy could be attributable to viable non-neuroblastic stromal cells. In this particular group of highly malignant NB tumors, new therapies addressing specifically the stromal compartment of tumor cells might be important. The observation that S-type cells may also be resistant to complement mediated cytotoxicity because of their surface expression of decay-accelerating factor (CD55), CD59, and CD45 additionally underscores the limitations of current antibody-based immunotherapy strategies (29) .

An interesting finding from our LSC analysis is that the widely accepted assumption in flow-cytometry, that the presence of a diploid peak in a hyperploid tumor reflects nontumoral origin (30) , may not always be true. The presence of small diploid GD2-positive clonal peaks in cases with predominant hyperploid clonal populations suggests that both are tumor derived. This and previous studies describing tumors with evolution of two clonal tumoral ploidy populations (31) suggest that the triploid clones may have arisen from diploid clones that had undergone endoduplication and subsequent triploidization as proposed by some investigators (32 , 33) .

In conclusion, our results provide evidence that neuroblasts and stromal cells in NB are of neoplastic origin and that the presence of stroma is a consequence (not a cause) of the maturation potential of tumor cells. These data support the hypothesis that NB arises from a tumoral neural crest cell capable of development along multiple lineages.

	ACKNOWLEDGMENTS

 
We thank Drs. Robert A. Ross, Barbara A. Spengler, and June Biedler for helpful discussions and review of the manuscript and Lishi Chen for his technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by the American Society of Clinical Oncology Young Investigator Award 2000 (to J. M.), the JP’s Wish Fund, the Katie-Find-a-Cure Fund, the Katie Hoch Foundation, and the Pediatric Cancer Foundation of New York.

² To whom requests for reprints should be addressed, at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-5905; Fax: (212) 639-4559; E-mail: geraldw{at}mskcc.org

³ The abbreviations used are: NB, neuroblastoma; SS, Schwannian stromal; LR, local regional; LCM, Laser-Capture Microdissection; LSC, Laser-Scanning Cytometry; FISH, fluorescence in situ hybridization; PI, propidium iodide; AI, allelic imbalance; LOH, loss of heterozygosity; FCM, flow cytometry.

Received 4/24/01. Accepted 7/16/01.

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