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Original Studies |
Clinical Biochemistry (C.C.R., M.L.B., M.P., C.O.), Andrology (M.M.), Gastroenterology (D.R., A.C.), and Endocrinology (M.S.) Units, Department of Clinical Physiopathology, and Department of Pediatrics (G.B.), University of Florence; Laboratory of Population Genetics, National Institute for Cancer Research (P.S., G.P.T.); and Department of Hematology-Oncology, Giannina Gaslini (B.D.B.), Genoa, Italy
Address all correspondence and requests for reprints to: Mario Maggi, M.D., Andrology Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. E-mail m.maggi{at}dfc.unifi.it
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We previously reported that several human NB cell lines expressed a rather variable density of receptors for the neuroendocrine hormone somatostatin (SS) (29). Later, we found that there was a dramatic correlation among the quantitative evaluation of sst2 protein expression (binding studies), sst2 gene expression (competitive RT-PCR), and SS responsiveness (17, 29). We found that only cell lines with a density of sst2 expression over 7 x 107 molecules/µg ribonucleic acid (RNA) were responsive to SS in terms of DNA synthesis, inhibition of adenylate cyclase activity, and calcium mobilization (17, 29). Hence, we suggested that this concentration of transcript represents a sort of biological threshold separating SS-responsive from unresponsive NB cells (17). By using competitive RT-PCR, we analyzed sst2 expression in a limited number of primary NB tumors (17). We essentially found that the abundance of sst2 transcripts in NB tumors was quite variable, spanning over 4 log orders of magnitude. Dividing NB tumors according to the aforementioned biological threshold of sst2 expression (7 x 107 molecules/µg RNA), we found that high levels of sst2 were positively related to survival (17, 30). A further study demonstrated that the ex vivo quantitative determination of sst2 was in good agreement with the in vivo semiquantitative determination of sst2 protein, as assessed by indium-111-pentetreotide imaging (30).
In the present report we have studied retrospectively 54 children with NB in whom sst2 expression was measured by competitive RT-PCR. This parameter was correlated to initial prognostic feature and patient outcome to determine its prognostic relevance and clinical impact.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We studied 54 tumors from children with NB that had been diagnosed from 1988 to 1997 in Italy. The only criterion for patient selection was the availability of sufficient amounts of RNA for quantitative PCR analysis. DNA was also available for all samples. Nucleic acid specimens were obtained from the Italian Neuroblastoma Tissue Bank (Genoa, Italy). Tissue samples were obtained at surgery and snap-frozen in liquid nitrogen. Total RNA and DNA were extracted from frozen samples with a standard phenol-chloroform procedure. In addition, each RNA extract was submitted to the treatment with 4 U RQ1 ribonuclease (RNase)-free deoxyribonuclease (Promega Corp. Italia, Milan, Italy).
All patients were staged according to the International Neuroblastoma Staging System (INSS) (31) as follows: 9 patients (16.7%) stage 1; 5 patients (9.3%) stage 2A; 4 patients (7.4%) stage 2B; 10 patients (18.4%) stage 3, 21 patients (38.9%) stage 4, and 5 patients (9.3%) stage 4S.
Competitive PCR for MYCN amplification and sst2 messenger RNA (mRNA) measurement
The number of MYCN copies in extracted DNA was measured with a competitive PCR assay using a cloned multiple competitor (pONC) containing competitive sequences for different oncogenes (MYCN, c-myc, int-2, c-erbB-2, and epidermal growth factor receptor) and for the single copy reference gene ß-globin (32). The primers and assay procedure were previously reported (32). The method included two subsequent PCR reactions. In the first, the exact amount of DNA was determined by referring to the ß-globin gene. At least three dilutions of pONC DNA (from 1500–6000 molecules) were mixed with a constant amount of extracted DNA (5 ng) and subjected to PCR amplification. PCR products, corresponding to DNA target and competitor template, were then resolved on a 12% polyacrylamide gel, stained with ethidium bromide, and quantitated by image analysis (33). The densitometric ratios of the two bands were plotted vs. the concentrations of added competitor. The exact number of DNA molecules in the initial sample was extrapolated at the equivalence (i.e. the competitor concentration that gives a 1:1 competitor/target ratio).
Oncogene amplification was measured for each sample in a second PCR, in which a constant amount of DNA (1,500 genome equivalents) was mixed with three dilutions of pONC competitor (1,500, 4,500, and 13,500 molecules), corresponding to MYCN amplifications of 1-, 3-, and 9-fold. For samples exceeding 9-fold amplification, a further PCR was performed, reducing the initial sample DNA concentration accordingly. The electrophoretic resolution of PCR products and image analysis calculation was the same as that previously described for ß-globin assay. PCR amplification of ß-globin and MYCN genes was performed with a Gene Amp 2400 thermal cycler (Perkin-Elmer Corp., Norwalk, CT) with a 40-cycle program at 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s.
Similarly, for the measurement of sst2 mRNA expression we
developed a synthetic RNA competitor cloned in pSSR-I plasmid
downstream to the T7 RNA polymerase promoter, as previously reported
(17). For each sample, 1 µg total RNA was mixed with an increasing
quantity of RNA competitor (generally from 1 x
105 to 1 x 109
molecules) and reverse transcribed in a 25-µL reaction volume
containing 2.5 µmol/L random examiners (Roche Molecular Biochemicals, Mannheim, Germany), 50 U Moloney leukemia virus
reverse transcriptase (Perkin-Elmer Corp.), 0.2 U RNase
inhibitor (Promega Corp.), 1 mmol/L deoxy-NTPs
(Roche Molecular Biochemicals), and 5 mmol/L
NaCl2. PCR amplification was performed with
described primers (17) with 40 cycles of 30 s at 94 C and 60
s at 62 C plus a final extension step for 7 min at 62 C. Each sample
was also submitted to a conventional PCR with the same primers and
cycling, but without RT, to exclude the presence of residual genomic
DNA in extracted specimens. The exact amount of sst2 mRNA
was calculated after densitometric analysis of 10 µL PCR products, as
described for MYCN measurement, and expressed as mRNA
molecules per µg total RNA. Figure 1
shows two typical competitive RT-PCRs for sst2 measurement
in a neuroblastoma tumor (A and B) and in normal human testis (C),
employed as an example of a low sst2-expressing tissue.
Figure 1B shows the linear relationship between the number of
competitor molecules added and the densitometric ratios between
products corresponding to competitor and transcript, as derived from
Fig. 1A. The number of sst2 mRNA molecules was extrapolated
from the point closer to the equivalence of a 1:1 competitor/target
ratio. Figure 1C shows experimental controls, including positive
controls, PCR blank, and analysis of a very low
sst2-expressing tissue.
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Frozen sections (7 µm thick) were collected onto gelatin/chrome alum-coated slides, dried briefly on a hot plate at 80 C, and fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), pH 7.4, for 20 min. After three washes in PBS and short air drying, sections were immediately used for in situ hybridization.
For preparation of the RNA probe for sst2, the 284-bp fragment of the human sst2 complementary DNA obtained by RT-PCR with the primers described was subcloned into the appropriate restriction site of the plasmid pGEM-T Vector (Promega Corp., Madison, WI). After linearization of the plasmid with either ApaI or SalI restriction endonuclease, SP6 or T7 RNA polymerase (Roche Molecular Biochemicals) was employed to obtain run-off transcripts of the antisense (complementary to mRNA) or sense (anticomplementary, negative control) strands, respectively. Transcription and labeling of RNA probes were performed as previously described (33, 34), using 60 µCi [35S]UTP (1250 Ci/mmol; NEN Life Science Products, Dreieich, Germany). RNA probes were stored at -80 C and used within 2 weeks. The specific activity routinely obtained was 1.2–1.4 x 109 cpm/µg.
Prehybridization, hybridization, removal of nonspecifically bound probe by RNase A digestion, and further washing procedures were performed for positive and negative strand RNA probes as described previously (34). After exposure for 4–6 weeks at 4 C, slides were developed using Kodak D19 developer (Kodak-Pathé, Chalon-S-Saône, France) for 3 min, rinsed in 1% acetic acid, and fixed in Kodak Unifix. After extensive washing, sections were counterstained with hematoxylin and eosin and mounted in Corbitt balsam.
Immunohistochemistry (IHC)
For IHC, frozen sections (7 µm) serial to those used for in situ hybridization were collected onto clean slides and air-dried overnight at room temperature. After fixation in 4% PFA/PBS, pH 7.4, for 20 min, sections were washed with PBS and incubated in normal goat serum diluted 1:10 in PBS/BSA 2% for 20 min. After two washes in PBS of 5 min each, sections were incubated in 1% H2O2/methanol to block endogenous peroxidase activity.
Immunolocalization of sst2 protein was performed using a polyclonal antiserum (SS-800, Gramsch Laboratories, Schwabhausen, Germany) diluted 1:400 in PBS/1% BSA. This antiserum was generated against the amino acid sequence 355–369 and recognizes the C-terminus of the human SS receptor type 2A. Immunohistochemical staining was then performed using the DAKO Corp. Envision+ System HRP (DAKO Corp., Carpinteria, CA), according to the manufacturer’s instructions. Finally, sections were weakly counterstained with Mayer’s hemalum and coverslipped using Kayser’s gelatin.
Negative controls were performed by omitting the primary antibody (to control the detection system) and employing nonimmune rabbit serum as the first layer. In addition, the specificity of the immunoreaction was verified by preabsorption of sst2 antiserum with its cognate receptor peptide.
Quantitative image analysis
Quantitative evaluation of sst2 mRNA and protein expression was performed by two independent observers who did not know the results of competitive RT-PCR, with the aid of a computerized video image analysis system (Quantimet Q500MC, Leica Corp. Cambridge Ltd., Cambridge, UK). For the quantitation of in situ hybridization results, six visual fields were chosen randomly from each section and analyzed under a darkfield microscope equipped with a x40 lens. The autoradiographic signal corresponding to the specific hybridization was acquired by a CCD video camera connected to the microscope, converted to digital, and transformed into pixel units. The threshold of specific detection was automatically calibrated on control sections hybridized with the corresponding sense probes. For quantitation of the immunohistochemical staining, six high power (x40) visual fields were chosen randomly from each section; the video image was generated again by the video camera and digitized for image analysis at 256 gray levels. An optical threshold and filter combination was set to select only the immunohistochemical signal.
The results, evaluated in terms of the percentage of the total area occupied by the sst2 autoradiographic and immunohistochemical signal, are expressed as the mean ± SD.
Statistical analysis
Statistical analyses were performed with software from SPSS, Inc. (Chicago, IL). The associations between clinical characteristics and molecular variables were tested with Fisher’s exact test. The probability of cumulative survival in various subgroups was tested according to Kaplan-Meier life tables (36). Survival differences between groups were tested with the log-rank method. Univariate and multivariate prognostic effects were evaluated with the Cox proportional hazards model (37). The comparison of sst2 expression levels among groups was performed using the Kruskal-Wallis test.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We found a wide range of expression of sst2 mRNA in all
NB analyzed (from 2.5 x 105 to 8 x
109 molecules/µg RNA). As shown in Fig. 2, a significant reduction of
sst2 expression was observed in patients in stage 4 compared
to other stages (P = 0.0092).
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The main clinical characteristics of the patient population and the
association of these features with sst2 mRNA levels are
shown in Table 1. Among the variables
tested, stage and MYCN amplification were significantly
related to sst2 expression (P < 0.02 and
P < 0.001, respectively). No relation was found
between sst2 expression, patient sex, and age at diagnosis
(P = 0.1635 and 0.11656, respectively).
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. Children with tumors showing high
sst2 expression had a significantly better survival rate
than patients with low sst2 expression (P <
0.00001; Fig. 3, upper panel). Similarly, a significant
inverse relationship was found between MYCN amplification
and patient survival (P = 0.0006; Fig. 3, lower
panel). Analysis of disease-free survival curves further
strengthened the high predictivity of sst2 detection
(P < 0.00001; Fig. 4, upper panel). Conversely, the evaluation of MYCN
amplification for disease-free survival was less informative, although
still retained a statistically significant predicting power
(P = 0.032; Fig. 4, lower panel).
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). The subgroup with low sst2
expression and MYCN amplification had the lowest survival
probability (22%). Overall, analysis of sst2 expression was
more predictive than and was independent from MYCN
amplification for mortality (P < 0.00001). Children
with high sst2 expression and MYCN amplification
showed a 75% survival probability. Conversely, the survival rate was
only 40% in children with low sst2 expression but without
MYCN amplification.
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), we
tested the prognostic value of sst2 expression and other
well known prognostic markers, such as tumor stage, age, and
MYCN amplification. In our patient population, all four
parameters had a significant prognostic value. To identify the most
powerful prognostic factors, we performed multivariate analysis with
the Cox proportional hazards model containing the aforementioned
prognostic parameters. According to the Cox regression model,
sst2 expression was the only independent prognostic factor
for the prediction of patient survival (P = 0.0149;
Table 2).
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In a subset of neuroblastoma samples (n = 7), sst2
mRNA expression was also evaluated by in situ hybridization,
and in five of them, receptor protein was immunolocalized in the
corresponding serial sections. Quantitative image analysis of serial
sections revealed an excellent correspondence between sst2
mRNA and protein expression at the tissue level (r = 0.978). We
also found that results from in situ hybridization and IHC
were well related to the expression of sst2 mRNA as detected
by competitive RT-PCR in the corresponding cancer specimens (RT-PCR vs
in situ, r = 0.756; RT-PCR vs. IHC, r =
0.814). Linear relationships are reported in Fig. 6. Figure 7
shows results obtained with the three aforementioned techniques in two
representative cases of neuroblastoma with different stages of disease
(stages 2A and 4). Quantitative RT-PCR indicated a very high level of
sst2 expression in the former (8 x
109 molecules/µg RNA) and a relatively low
expression in the latter (1 x 107
molecules/µg RNA). Accordingly, the positive signal for either
sst2 gene or protein was definitively more abundant in the
neoplastic tissue from stage 2A than in that from stage 4.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Somatostatin is a peptide that, aside from its neuroendocrine functions, also regulates cell proliferation. Recently, a family of SS receptors, essentially characterized by different affinities for several SS analogs, has been cloned (38). Among the different SS receptor subtypes, sst2 is the most sensitive to the commercially available analogs (38) and the most represented in neuroendocrine tumors (39, 40, 41). In addition, sst2 mediate, at least in part the antiproliferative activity of SS. Using an original competitive RT-PCR assay, we demonstrated that sst2 is variably expressed in NB cell lines and that this expression is highly correlated to cellular responsiveness to SS in vitro (17).
In the present study we measured the specific transcript for this receptor subtype in 54 NB. The level of sst2 gene expression was variable, spanning more than 4 log orders of magnitude. Although a high concentration of receptors was detected in NB tumors belonging to all different clinical stages, the lowest level of sst2 was found in the most aggressive stage of the disease (stage 4). In the more favorable stages (stages 1, 2A, and 2B) children expressed elevated concentrations of sst2, with the exception of one child who showed low expression of sst2 and died of the disease. The large majority of children (88%) classified in unfavorable stage 3 or 4 but expressing an elevated density of receptors are still alive. Hence, we hypothesized that the main clinical relevance of sst2 measurement in NB lies in its ability to detect patients at low risk for poor outcome or for recurrent disease.
Analysis of overall and disease-free survival curves according to sst2 expression strongly suggested that this is the case. When the outcome was adjusted to the effect of MYCN, sst2 mRNA expression remained significant. It is important to note that high sst2 expression and a single MYCN copy number identify in our study population a subgroup of children with a totally favorable outcome (100% survival). Of greater importance are results from multivariate analysis. When several established prognostic indicators were combined with sst2 expression as variables in the Cox regression model, only sst2 retained its prognostic significance, independently from all other prognostic indicators.
In this study we also confirmed a previous result (17) indicating a good correspondence between different methods of sst2 mRNA detection in NB samples. Indeed, we previously reported similar results with Northern analysis and competitive RT-PCR (17). In the present study we found a good agreement between the latter method and quantitative analysis of in situ hybridization. More interestingly, we also found a positive relationship between in situ hybridization and IHC results for sst2 in serial sections of the same tumors. In addition, the quantitative image analysis of these preparations revealed good relationships between the measurements with morphological techniques and those obtained with competitive RT-PCR. This is in keeping with previous results showing that the levels of sst2 mRNA expression, measured with competitive RT-PCR, were significantly related to the in vivo semiquantitative determination of sst2 protein, as assessed by indium-111-pentetreotide imaging (30). Finally, we previously demonstrated that the sst2 mRNA concentration was highly correlated to the level of biologically active sst2 protein, as evaluated by binding and functional studies of human neuroblastoma cell lines (17, 29). This evidence demonstrates that in neuroblastoma quantitative determination of sst2 mRNA is highly related to expression of the relative protein.
In conclusion, we demonstrated that quantitation of sst2 expression can provide relevant prognostic information, giving further insight into the biological features of NB. The measurement of this parameter at the time of diagnosis could help to determine the appropriate choice and intensity of treatment. Again, the main clinical value of sst2 as a prognostic marker lies in its ability to detect patients at low risk, independently from the clinical stage, age of diagnosis, and MYCN amplification. Although there is some suggestion that therapy with SS or analogs might provide benefits in cancer patients, this does not seem to be the case in NB. Indeed, according to the present data, patients with high expression of sst2 (and therefore sensitive to SS therapy) have per se a quite favorable outcome. Hence, adjuvant therapy with SS is not necessary. Conversely, patients with poor outcome are essentially characterized by low expression of sst2 (and therefore insensitive to SS therapy). In this case adjuvant therapy with SS might be indicated, but would have few chances of success. In addition, a recent report (42) indicates that the more differentiated NB tumors express a high concentration of SS. It is possible that the degree of expression of SS regulates sst2 expression, because sst2 is known to undergo agonist-induced down-regulation (43). NB cells expressing an elevated density of sst2 receptors (and its natural ligand) may proliferate less (leading to tumor regression) or differentiate more (leading to benign ganglioneuroma) than cells with low expression. Therefore, we believe that induction of sst2 gene expression by medical therapy (i.e. steroids) (44) or gene therapy (45) is a more appropriate goal for the future than the conventional administration of SS and its analogs.
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Footnotes |
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Received October 11, 1999.
Revised June 1, 2000.
Accepted June 15, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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