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Clinical Cancer Research Vol. 6, 551-558, February 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Expression of Protein Gene Product 9.5 and Tyrosine Hydroxylase in Childhood Small Round Cell Tumors1

Yinglin Wang, Peggy Einhorn, Timothy J. Triche, Robert C. Seeger and C. Patrick Reynolds2

Division of Hematology-Oncology, Childrens Hospital Los Angeles, Los Angeles, California 90027 [Y. W., P. E., R. C. S., C. P. R.], and Departments of Pediatrics [R. C. S., C. P. R.] and Pathology [Y. W., T. J. T., C. P. R.], University of Southern California School of Medicine, Los Angeles, California 90033


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small round cell tumors of childhood can be histologically ambiguous, can require tumor markers for an accurate diagnosis, and include neuroblastoma, peripheral primitive neuroectodermal tumor (pPNET), Ewing’s sarcoma (ES), lymphoma, and rhabdomyosarcoma. Because the cell type of origin for ES remains controversial, characterizing gene expression in ES can provide diagnostic markers and lead to better understanding of tumor biology. We studied RNA expression of the neuronal genes protein gene product 9.5 (PGP 9.5) and tyrosine hydroxylase (TH) by Northern analysis in cell lines and tissue from small round cell tumors. PGP 9.5 showed strong expression in 17 of 17 neuroblastoma cell lines, 9 of 9 pPNET cell lines, and 11 of 11 ES cell lines. PGP 9.5 was weakly expressed in 1 of 1 alveolar rhabdomyosarcoma cell lines but not in 1 of 1 embryonal rhabdomyosarcomas, and weak expression was seen in 1 of 7 leukemia cell lines. In tumor tissue, all 12 neuroblastomas expressed PGP 9.5, as did all 7 pPNET and all 7 ES. PGP 9.5 was very weakly expressed in 6 of 9 rhabdomyosarcomas and 1 of 9 lymphomas. TH was expressed only in neuroblastomas, and no TH expression was seen in cell lines or tissue from other tumors. As high expression of PGP 9.5 was only found in neural tumors; PGP 9.5 expression by ES provides further evidence for a neural origin of this tumor, whereas TH expression is highly specific for neuroblastomas. PGP 9.5 expression should allow sensitive detection of minimal residual disease for ES and pPNET using reverse transcription-PCR, and the variability in TH and PGP 9.5 expression levels in neuroblastomas indicates that expression of both genes should be used for monitoring minimal residual disease by reverse transcription-PCR.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small round cell tumors of childhood, which conventionally include NB,3 ES, and the genetically related pPNET, lymphoma, and rhabdomyosarcoma (largely the solid alveolar variant), can be nearly isomorphous on histological examination and often require detection of various tumor markers for accurate diagnosis (1) .

The cell of origin for ES and pPNET remains unproven, with cell types such as endothelial (2) , vascular pericytes or smooth muscle (3) , and multipotential mesenchymal (4) cells all having been suggested in the past. The reciprocal translocation t(11;22)(q24;q12) (Ref. 5 ) was present in ~90% of ES/pPNET cases (6) , and this suggested that like pPNET, ES is of neuroectodermal origin. That unique translocation results in formation of the EWS/FLI-1 fusion protein (7) , and the translocation (or its gene product) provides a fairly reliable marker for the diagnosis of ES/pPNET (8 , 9) . The presence on ES of neuroectodermal epitopes recognized by the HNK-1 antibody (10) , the demonstration of a neuroectodermal ultrastructural nature in vitro (11) , and induced neural differentiation of ES cell lines (12) also support a neural histogenesis for ES. Moreover, tumor cells of both ES and pPNET showed high expression of the pseudoautosomal p30–32 MIC2 antigen (13) , providing further evidence that ES and pPNET tumors are likely closely related. Characterizing patterns of gene expression in ES and pPNET can provide diagnostic markers and also a better understanding of the biology of this enigmatic tumor.

We studied the expression of two neuronal genes (PGP 9.5 and TH) in cell lines and tumor tissue from small round cell tumors of childhood. PGP 9.5 was discovered as a human "brain-specific" protein by high-resolution, two-dimensional electrophoresis of human brain soluble proteins (14 , 15) . It is expressed specifically in the cytoplasm of almost all neurons at a concentration similar to, or higher than, NSE, and it comprises 1–5% of total soluble brain protein (14) . Immunohistochemistry has shown that PGP 9.5 protein is expressed only in neurons of both the central and peripheral nervous systems as well as in neuroendocrine cells and tissues (14, 15, 16) . The cloning and sequencing of the gene has led to the discovery of PGP 9.5 to be identical to ubiquitin carboxyl terminal hydrolase 1, a neuron-specific protein involved in the ubiquitin-mediated proteolytic pathway (17) .

TH is the first and rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). TH is expressed in catecholaminergic neurons in discrete regions of the brain, in noradrenergic neurons of sympathetic ganglia in sympathetic nerves, and in the adrenal medulla (18) . On the basis of excretion of metabolites in urine, 95% of neuroblastomas synthesize catecholamines (19) , and catecholamine-negative neuroblastomas have TH activity (20) , suggesting that TH may be a specific and sensitive marker for NB. Because other small round cell tumors are catecholamine negative (21) , and TH activity is negative in ES and pPNET cell lines (22) , TH may be a specific marker for NB among small round cell tumors.

Using Northern blot analysis, we measured PGP 9.5 and TH RNA expression in a series of well-characterized cell lines and in tumor tissue from small round cell tumors of childhood, and we compared the relative expression of these two genes among the different categories of tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Primary Tumors.
A panel of 47 tumor cell lines was analyzed in this study. Their diagnoses and references are listed in Table 1Citation (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) . Cell lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum at 37°C in a 5% CO2 atmosphere, without antibiotics, and were free of Mycoplasma.


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  		Table 1 Relative PGP 9.5 and TH RNA expression in small round cell tumor cell lines

 
Tissue from 44 tumor samples was also examined, and the clinical parameters for these cases are listed in Table 2Citation . Tumor samples were frozen within 1 h of surgical removal and stored at -70°C. Tumor cellularity was verified for each tumor sample by embedding a portion of tumor adjacent to the tissue used for RNA extraction in Tissue-Tek OCT compound, and pilot frozen sections were made and stained with H&E. Only highly cellular and viable portions of tumor were used for RNA extraction. A bone marrow sample harvested for autologous transplantation was obtained from a patient with a brain tumor.


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  		Table 2 Relative PGP 9.5 and TH RNA expression in small round cell tumor tissue

 
RNA Extraction and Northern Blot Analysis.
RNA was extracted from cell lines using the acid-guanidinium-phenol-chloroform method (46) . RNA from tumors was extracted from frozen tissue using TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD), and 20 µg of RNA of each sample were electrophoresed through a 1.1% agarose/formaldehyde gel in 4-morpholinepropanesulfonic acid X1 buffer, transferred to nylon membrane (Bio-Rad, Hercules, CA), and cross-linked by a UV-Stratalinker (Stratagene, La Jolla, CA). The membranes were first prehybridized at 65°C in prehybridization solution [1 mM EDTA, 0.5 M Na2HPO4 (pH 7.2), and 7% SDS] for 1 h, and then hybridized at 65°C with the same buffer containing 32P-labeled cDNA probes prepared with a random-primed labeling kit (Boehringer Mannheim, Mannheim, Germany) for 16 h. After 16 h of hybridization, membranes were washed at 65°C three times for 30 min in wash buffer [1 mM EDTA, 40 mM Na2HPO4 (pH 7.2), and 5% SDS]. The cDNA probes used were human PGP 9.5, generously provided by Dr. Ian Day (47) , TH, kindly provided by Dr. Toshiharu Nagatsu (18) , and human ß-actin (Life Technologies). Blots were stripped each time between hybridization by washing two times, 20 min each, in a large volume of 0.1x SSC/0.5% SDS at 95°C so that one blot was used for all three cDNA probes. Blots were exposed to Hyperfilm-MP (Amersham, Arlington Heights, IL) with intensifying screens at -80°C for 3–6 days, depending on the intensity of the signals.

Molecular Imager and Sigma Plot Analysis.
The same set of blots was used for PGP 9.5, TH, and ß-actin hybridization after probe stripping. Each time, the hybridized membranes were exposed to a molecular imaging screen, and the signals were quantified by the Phosphor Analyst/PC (Bio-Rad). The quantity of PGP 9.5 and TH expression was normalized to ß-actin from the same blot to adjust for the amount of RNA loaded. The highest expressing cell lines were used to set a value of 100 (NMB for PGP 9.5 and LA-N-6 for TH). The relative expression levels from different blots were calculated relative to a standard cell line (SMS-SAN) on each blot. The data were analyzed and graphed using SigmaPlot 4.0 (Jandell Scientific, San Rafael, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PGP 9.5 Expression in Cell Lines.
As shown in Table 1Citation , a total of 47 small round cell tumor cell lines were examined for expression of PGP 9.5. Fig. 1ACitation shows representative Northern blots. The relative expression for each cell line is shown by the graph below the blot with each column on the graph corresponding to each lane on the blot. PGP 9.5 was expressed as a 1-kb transcript. The highest expressing cell line (NMB) was defined as 100, and the expression level of PGP 9.5 was divided into four categories: high 50–100(50–100), intermediate 10–50(10–50), low (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) , and no expression (0). Fig. 1BCitation summarizes the expression levels of PGP 9.5 in all of the small round cell tumor cell lines tested, and the relative expression values of PGP 9.5 for all 47 cell lines are listed in Table 1Citation . All NBs expressed PGP 9.5; 6 of 17 expressed at high levels, and 11 of 17 expressed at an intermediate level. PGP 9.5 was detected in all 9 pPNET cell lines; 1 of 9 expressed at high level, 7 of 9 expressed at an intermediate level, and 1 of 9 expressed at low level. For ES, all 11 cell lines expressed PGP 9.5, and 3 of 11 expressed at a high level, 6 of 11 expressed at an intermediate level, whereas 2 of 11 showed low level expression. PGP 9.5 was not expressed in 1 of 1 embryonal rhabdomyosarcomas, but 1 of 1 alveolar rhabdomyosarcoma cell line showed an intermediate level of PGP 9.5 expression (Fig. 1Citation B, Lane 15). Seven leukemia cell lines were analyzed, and PGP 9.5 was not expressed in six of the leukemia lines, but weak expression was detected in the SMS-SB pre-B leukemia cell line.



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  		Fig. 1. PGP 9.5 RNA expression in small round cell tumor cell lines. A, representative Northern blots. Total RNA was extracted from cell lines and subjected to Northern analysis with 32P-labeled PGP 9.5. Blots were stripped and reprobed with ß-actin cDNA to normalize the loading of RNA. The corresponding column on the graph shows the relative expression for each cell line (highest expression defined as 100). NB: Lane 1, LA-N-5; Lane 2, LA-N-6; Lane 3, SK-N-AS; Lane 4, NMB; Lane 5, SK-N-DZ. pPNET: Lane 6, CHP-100; Lane 7, SK-N-DW; Lane 8, SK-N-LO; Lane 9, SK-N-MC. ES: Lane 10, A9423; Lane 11, CHP-196; Lane 12, N-1001; Lane 13, N-1002. Rhabdomyosarcoma (RMS): Lane 14, CB-NJR; Lane 15, RD. Leukemia (Leuk): Lane 16, MOLT-3; Lane 17, NALL-1; Lane 18, NALM-6; Lane 19, SMS-SB. B, a composite graph showing the levels of PGP 9.5 expression in cell lines from small round cell tumors. Each triangle represents a sample. The median levels of expression for each tumor type are depicted by the heavy horizontal bars. Abbreviations used are the same as in A. The number of the samples analyzed is indicated under each tumor type.

 
The median expression levels of PGP 9.5 for NB, pPNET, ES, rhabdomyosarcoma, and leukemia cell lines were 37, 29, 21, 6.5, and 0, respectively (shown by the heavy horizontal bars in Fig. 1B)Citation . A Kruskal-Wallis nonparametric test was used to analyze the intergroup differences. PGP 9.5 expression in neural tumors (NB, pPNET, and ES) was significantly higher than that in nonneural tumors (rhabdomyosarcoma and leukemia) at an overall {alpha} level of 0.05. Within the neural tumors, the differences of expression were not significant.

PGP 9.5 Expression in Tumor Tissue.
As shown in Table 2Citation , we measured PGP 9.5 RNA expression in tumor tissue from 12 neuroblastomas, 7 pPNETs, 7 ESs, 9 rhabdomyosarcomas, and 9 lymphomas. Clinical data for these cases and relative PGP 9.5 expression levels are listed in Table 1Citation . A tumor-free bone marrow was used as a negative control. Representative Northern blots are shown in Fig. 2Citation A, with the graph showing the relative expression normalized to ß-actin. The expression levels of PGP 9.5 in tumor tissue were calculated relative to that of cell lines, also with the highest expression in cell line NMB defined as 100. Fig. 2BCitation summarizes the level of PGP 9.5 RNA expression for all of the tumor tissues studied. PGP 9.5 was expressed in all tumor tissues from NBs, pPNETs, and ESs. For NBs, 10 of 12 expressed PGP 9.5 at a high level 50–100(50–100), and 2 of 12 expressed PGP 9.5 at an intermediate level 10–50(10–50). No expression was detected, even with an exposure time of 10 days in the normal bone marrow sample (Fig. 2Citation A, Lane BM). Only 1 of 7 pPNETs expressed PGP 9.5 at a high level 50–100(50–100), 5 of 7 showed intermediate levels of expression 10–50(10–50), and 1 of 7 at a low level (2.4). For ES tumor tissue, 4 of 7 expressed PGP 9.5 at an intermediate level 10–50(10–50), and 3 of 7 expressed PGP 9.5 at a low level (5, 6, 7, 8, 9, 10) . PGP 9.5 was expressed very weakly in 6 of 9 rhabdomyosarcomas (level <6) and was not expressed in 3 of 9 rhabdomyosarcomas. Examining the subtypes of rhabdomyosarcoma showed that 4 of 4 alveolar tumors expressed PGP 9.5, as did 2 of 3 embryonal tumors, whereas no PGP 9.5 expression was seen in 1 of 3 embryonal, 1 of 1 botryoid, and 1 of 1 mixed alveolar/embryonal rhabdomyosarcomas. Very weak expression of PGP 9.5 was seen in 1 of 9 lymphomas (level <1), whereas no expression was detected in the other 8 of 9 lymphomas.



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  		Fig. 2. PGP 9.5 RNA expression from small round cell tumor tissue. A, representative Northern blots. Blots were hybridized with PGP 9.5 and then ß-actin cDNA probes as indicated. Each lane of the Northern blot corresponds to the column on the graph below showing relative expression of PGP 9.5 (normalized to ß-actin). NB: Lane 1, NB1; Lanes 2–4, NB9–11. pPNET: Lanes 5–8, PNET2–5; ES: Lanes 9–12, ES3–6. Rhabdomyosarcoma (RMS): Lane 13–16, RMS1, RMS2, RMS6, RMS7. Lymphoma (Lymph): Lanes 17–20, Lymph1–4; Lane BM, bone marrow. B, a composite graph showing PGP 9.5 expression in tumor tissues from small round cell tumors. Each triangle depicts a sample. The median values of expression for each tumor type are represented by the heavy horizontal bars. Abbreviations are the same as in A. Sample size is shown under each tumor type.

 
The median values of expression were 74, 19, 10, 1.3, and 0 for NB, pPNET, ES, rhabdomyosarcoma, and lymphoma tissue, respectively (shown by the heavy horizontal bars in Fig. 2B)Citation . The intergroup variances were analyzed using a Kruskal-Wallis nonparametric test. Expression of PGP 9.5 in neural tumors (NB, pPNET, ES) was significantly higher than that in nonneural tumors at an overall {alpha} level of 0.05. Within the group of neural tumors, PGP 9.5 expression was significantly higher in NB than in pPNET or ES tumors, whereas the difference between pPNET and ES was not significant.

TH Expression in Small Round Cell Tumors.
The same blots (both cell lines and primary tumors) were stripped after PGP 9.5 detection and were then hybridized with a 32P-labeled TH cDNA probe. TH was expressed as an ~1.9-kb transcript. Alternative splicing of TH from a single primary transcript generates four forms of mRNAs with slightly different sizes (48) , which may account for the wide appearance of the signal on the Northern blots. The expression levels for the cell lines are shown in Table 1Citation , for the tissue specimens in Table 2Citation , and representative Northern blots for NBs are shown in Fig. 3ACitation . Relative expression for those blots is shown on the graph below the blot. The TH expression among these samples was quantified and normalized to ß-actin, and the highest expression seen in the cell line LA-N-6 was defined as 100. Fig. 3BCitation displays the level of TH RNA expression in NB cell lines versus tumor tissue. TH was expressed in all 17 NB cell lines, but expression was highly variable from line to line. Low TH expression (<5) was seen in 6 of 17 NB cell lines. All 12 NB tissue specimens expressed TH, and the expression levels in tissue samples were more closely distributed. The median expression levels were 11.1 for cell lines and 15.5 for tumor tissues; however, the difference of expression between cell lines and tumor tissue was not statistically significant. No TH expression was detected in the normal bone marrow sample (Fig. 3Citation A, Lane BM).



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  		Fig. 3. Northern blot analysis showing TH expression in neuroblastoma cell lines and primary tumor tissue. The same sets of membranes stripped after PGP 9.5 detection were hybridized with 32P-labeled TH cDNA probe. A, NB cell lines. Lane 1, CHP-134; Lane 2, CHP-234; Lane 3, LA-N-5; Lane 4, LA-N-6; Lane 5, SK-N-FI; Lane 6, SK-N-RA; Lane 7, SK-N-SH; Lane 8, SMS-KAN; Lane 9, SMS-KCNR. Primary NB tumor tissues: Lanes 10–17, NB1–8; Lane BM, bone marrow. B, a composite graph showing TH expression in NB cell lines and tumor tissue. Each sample tested is represented by a triangle. The median expression levels are shown as the heavy horizontal bars. The number of the samples tested are indicated below the graph.

 
TH was not detected in any of the 30 non-NB lines or 32 non-NB tumor tissues, including pPNETs, ESs, leukemias, lymphomas, and rhabdomyosarcomas (Tables 1Citation and 2)Citation , even with a prolonged exposure time for the blots (data not shown). Integrity of RNA was confirmed by probing the same blots with ß-actin.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Differential diagnosis of small round cell tumors can be a challenge for clinicians and pathologists. Although careful gross examination and light microscopy in conjunction with clinical information can provide sufficient evidence for the diagnosis of most of tumors, differential diagnosis of certain cases are greatly dependent on the application of a host of additional diagnostic techniques.

We have evaluated the relative amounts of RNA expression for PGP 9.5 and TH genes in the small round cell tumors by Northern blot analysis in 47 cell lines and 44 tumors. PGP 9.5 was expressed strongly in most cell lines of neural origin tumors, such as NB, pPNET, and ES, but was not expressed or was expressed only weakly in most nonneural tumors such as lymphoma/leukemia and rhabdomyosarcoma. Similar results were obtained from tumor tissue, with PGP 9.5 being strongly expressed in NB, expressed intermediately in pPNET and ES, and expressed only very weakly in some of the rhabdomyosarcomas and lymphomas. Neither PGP 9.5 nor TH showed detectable expression in bone marrow.

Strong expression of PGP 9.5 by all ES cell lines and tumor tissue provides further evidence for a neural origin of this tumor. This also supports the well-accepted notion that ES and pPNET belong to the same clinicopathological entity of bone and soft tissue tumors, with typical undifferentiated ES lying at one end of a spectrum and pPNET with clear evidence of neural differentiation at the other (1) . The overall expression of PGP 9.5 in ES/pPNET tumors was weaker than that seen for NB, both in cell lines and tumor tissue. This is probably attributable to the lower level of differentiation of ES/pPNET along the neural lineage, which suggests that PGP 9.5 may relate to the degree of neuronal differentiation, similar to NSE (49) . The median level of expression for pPNET was higher than that for ES, although the difference was not statistically significant.

The very weak expression of PGP 9.5 by 1 of 7 leukemia cell lines (SMS-SB) and 1 of 9 lymphoma tissues (Table 2Citation , Lymph4) is not understood, but inappropriate gene expression is not uncommon in malignant cells. We saw weak PGP 9.5 expression in 2 of 3 embryonal rhabdomyosarcoma tumor samples. Although 1 of 1 embryonal rhabdomyosarcoma cell line did not express PGP 9.5, whereas 1 of 1 alveolar rhabdomyosarcoma cell line did express the gene, because of the small sample size, we are unable to draw any conclusions concerning expression of PGP 9.5 among the subtypes of rhabdomyosarcomas. The weak expression of PGP 9.5 in 1 of 2 rhabdomyosarcoma cell lines and 6 of 10 tissue specimens may be similar to the weak, positive immunohistochemical staining of NSE seen in rhabdomyosarcomas (50) . Some of tumors that were initially diagnosed as alveolar or primitive rhabdomyosarcomas on the basis of morphological and immunophenotypic features have later been shown to be biphenotypic sarcomas having both myogenic and neural phenotypes (51) . Such a biphenotypic form may explain the weak PGP 9.5 expression in rhabdomyosarcomas. Although not exclusively expressed in neural tumors, if used in combination with measuring expression of other genes, PGP 9.5 should be a useful member of a panel of markers for molecular diagnosis, and high expression of PGP 9.5 is limited to neural tumors.

Alternative splicing of TH from a single primary transcript generates four forms of mRNAs with slightly different size (48) . All these mRNA forms and protein isoforms have been identified in NB cells (52) ; however, the functional implications of the four isoforms are not yet clear. Using Northern blot analysis, we were able to detect TH RNA in all of the NB cell lines and tumor tissue. By contrast, no TH RNA was detected in any other members of the small round cell tumors including pPNET, ES, lymphoma/leukemia, and rhabdomyosarcoma, nor in the bone marrow sample. Thus, TH was expressed in a tumor type known to synthesize catecholamines, i.e., NB. Some NBs do not produce catecholamines that are detected either in the patient urine or in tumor tissue (21 , 53) . Some NB cell lines in this study were from tumors that failed to show catecholamine production in the patient (54) yet were clearly NB by other markers, i.e., neurite outgrowth in vitro (53) , cell surface antigens (29) , and patterns of insulin-like growth factor expression (55) . In this study, we showed that those cell lines (i.e., SMS-KAN and SMS-KCNR) were strongly positive for TH expression. Thus, TH may be a more sensitive diagnostic marker for NB than production of catecholamines.

NB, pPNET, and ES are all malignant tumors that frequently metastasize. Sensitive and specific methods for detecting small numbers of tumor cells in bone marrow or blood are needed to monitor minimal residual disease in patients and also to assess the potential for infusing tumor cells during autologous bone marrow or peripheral blood stem cell transplantation. Immunodetection of blood and marrow metastasis from NB using monoclonal antibodies directed against tumor surface antigens has been developed (56 , 57) . RT-PCR for both PGP 9.5 and TH expression has been used for the sensitive detection of occult neuroblastoma cells in bone marrow and peripheral blood (58, 59, 60, 61) . RT-PCR for EWS/FLI-1 fusion transcripts resulting from t(11;22)(q24;q12) has enabled the detection of ES/pPNET tumor cells in blood and bone marrow (61 , 62) . Because PGP 9.5 is expressed in pPNET and ES, RT-PCR for PGP 9.5 should also provide a useful method for determining minimal residual disease in these tumors, as it has for NBs, especially when used in conjunction with other markers, such as RT-PCR for EWS/FLI-1.

Determining the level of expression among a panel of cell lines or tumor tissue for PGP 9.5 or TH has not been done, and relative expression levels may affect the sensitivity of detection by RT-PCR. The more variable expression of TH RNA in NB suggests that the sensitivity for detecting NB with TH RT-PCR may vary from tumor to tumor, and this issue warrants further investigation. Moreover, because high expression of at least one of the two genes (PGP 9.5 or TH) was seen in most NB cell lines and tissue, both markers should be used for detecting minimal residual disease in NB.


   ACKNOWLEDGMENTS
 
We thank Drs. June Biedler, R. Graham Smith, Garett Brodeur, Audrey Evans, and Mark A. Israel for providing cell lines used in this study. We also thank Juan-Juan Zuo for technical assistance. We are grateful to Peter K. Wakamatsu for help in the statistical analysis.


   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 by research grants from the Neil Bogart Memorial Laboratories of the T. J. Martell Foundation for Leukemia, Cancer, and AIDS Research and The American Institute for Cancer Research, and supported in part by Grants CA13539 and CA60104 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at Division of Hematology-Oncology, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, MS#57, Los Angeles, CA 90027. Phone: (323) 669-5646; Fax: (323) 664-9455; E-mail: cpreynol{at}hsc.usc.edu Back

3 The abbreviations used are: NB, neuroblastoma; ES, Ewing’s sarcoma; pPNET, peripheral primitive neuroectodermal tumor; TH, tyrosine hydroxylase; PGP, protein gene product; NSE, neuron-specific enolase; RT-PCR, reverse transcription. Back

Received 8/31/99; revised 11/ 8/99; accepted 11/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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