ARTICLE |
Correspondence to: Giuseppe Pelosi, Divisione di Anatomia Patologica e Medicina di Laboratorio, Istituto Europeo di Oncologia, Via G. Ripamonti 435, I-20141 Milano, Italy.
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Summary |
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Recent reports have suggested that the immunodetection of tumor cells in bone marrow of small-cell lung cancer (SCLC) patients is by far more effective than traditional cytohistological methods and that this may be clinically relevant. This study aimed to evaluate whether the level of detection of tumor cells in bone marrow is affected by different immunostaining methods. Using two anti-NCAM monoclonal antibodies (MAbs), we compared four different "sandwich" methods on cytospin preparations of the N592 human SCLC cell line and of bone marrow aspirates from 37 SCLC patients. Our data indicate that the combination of the alkaline phosphataseanti-alkaline phosphatase and streptavidinbiotinalkaline phosphatase complex methods provides the best results in terms of sensitivity and specificity, and of intensity of immunoreaction and absence of staining background. Moreover, bone marrow micrometastases detected by this method were prognostically relevant and identified, among patients with apparently limited disease according to conventional staging procedures, a subgroup with shorter survival. We suggest that the choice of a sensitive immunostaining technique may significantly increase the detection rate of SCLC cells in bone marrow, mirroring the biological aggressiveness of the disease. (J Histochem Cytochem 47:10751087, 1999)
Key Words: small-cell lung cancer, bone marrow, immunocytochemistry, alkaline phosphatase, amplification, prognosis
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Introduction |
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Small-cell lung cancer (SCLC) is a highly aggressive tumor, and most patients have widespread disease at the time of diagnosis. Evidence is accumulating not only that bone marrow microcontamination by SCLC cells is by far more common than assessed by traditional cytohistological methods (
Many studies on the detection of SCLC micrometastases in BMA have employed antibodies to NCAM (
A certain number of studies addressing the localization of SCLC micrometastases in BMA by use of anti-NCAM antibodied have used standard methods of immunostaining (
This study compares four immunocytochemical staining "sandwich" methods to improve the detection rate of BMA micrometastases. We evaluated the immunoreactivity of two anti-NCAM antibodies in cytospin preparations of the N592 human SCLC cell line and of BMA from 37 SCLC patients. Moreover, we correlated the findings of bone marrow positivity with the clinicopathological features of the patients. Our study suggests that the choice of the immunocytochemical technique may affect the detection rate of metastatic tumor cells with anti-NCAM antibodies and that bone marrow micrometastases parallel the biological aggressiveness of the disease.
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Materials and Methods |
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Strategy of Study, Selection of Patients and Adequacy of Samples
We designed and conducted a two-phase experiment to test four different nonlabeled antibody detection methods based on alkaline phosphatase (AP) with two purified mouse anti-human NCAM MAbs to examine the effectiveness of different labeling conditions on the detection of contaminating tumor cells in BMAs of SCLC patients. In the first phase, we tested two antibodies, clone NCC-LU243 (IgG2a) and clone NCC-LU-246 (IgG1) (Nippon Kayaku; Tokyo, Japan) on cytospin preparations of the N592 human SCLC cell line using the following detection systems: APanti-AP (APAAP) technique; APAAPdouble bridge technique; combination of APAAP and streptavidinbiotinAP (SABAP) complex techniques, as described by
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Cell Line
The N592 human cell line, kindly provided by Dr. Sylvie Menard (National Cancer Institute; Milan, Italy), was grown in RPMI medium 1640 (MA Bioproducts; Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum and 80 mg/ml gentamycin at 37C in a fully humidified atmosphere of 5% CO2. Before use, the cultures were washed repeatedly in RPMI 1640 with 5% heat-inactivated fetal calf serum, and viable cells were counted by trypan blue exclusion at a final concentration of 11.5 x 106 cells/ml. Cytospin slides were then prepared for immunostaining using the same procedure as for BMA.
Bone Marrow Aspirates
All BMAs (35 ml each) were collected in disposable heparinized syringes, sedimented onto a FicollHypaque (Pharmacia; Uppsala, Sweden) density gradient, cytospun, and fixed in acetone. Briefly, after density centrifugation (400 x g for 30 min at 19C), the mononuclear cell layer was washed three times in RPMI medium 1640 supplemented with 15% heat-inactivated fetal calf serum (350 x g for 7 min at 4C) and then resuspended at 11.5 x 106 cells/ml. Then the isolated mononuclear cells were cytospun (Cytospin 3; Shandon Scientific, Cheshire, UK) by introducing 75 µl of the cell suspension into each sample chamber (Cytofunnel) and centrifuging them at 500 rpm for 4 min at room temperature (RT) on 3-aminopropyltriethoxysilane-coated slides (Sigma Chemical; St Louis, MO). According to this procedure, a monolayered spot of 0.6 cm diameter containing 57.5 x 104 well-preserved mononuclear cells was obtained for each slide. Cytospins were then fixed in pure acetone for 5 min at 4C and stored at -20 or -80C in aluminum foil. Before immunostaining, frozen cytospin preparations were air-dried for 1 hr and rehydrated in Tris buffer with 5% human serum.
Immunocytochemistry
Both primary antibodies were used at a concentration of 20 µg/ml at RT for 1 hr. Briefly, after the primary antibody incubation in the APAAP technique, polyvalent rabbit anti-mouse immunoglobulin and APAAP mouse complex were applied in sequence. In the APAAPdouble bridge technique, the second and third incubations of the previous procedure were repeated in the same sequence. In the combined technique, polyvalent biotinylated horse anti-mouse immunoglobulin, APAAP mouse complex, and again biotinylated horse anti-mouse immunoglobulin were applied in sequence and followed by labeling with the streptavidinAP complex (Figure 1). Endogenous AP activity was blocked with 1% w/v levamisole (Sigma) in the chromogen solution. AP activity was developed with 2% New Fuchsin (Merck; Darmstadt, Germany) and 0.2% naphthol AS-BI phosphate (Sigma) in propandiol buffer, pH 89 to yield a red endproduct. Slides were lightly counterstained with 1% Harris' hematoxylin. The specificity of all reactions was verified by replacing primary antibodies with unrelated mouse IgG in buffer at a comparable dilution. Cytospins of N592 cell line stained in parallel were employed as external positive control, and myeloid cells of each BMA were used as internal negative control. Moreover, BMAs from healthy volunteers and patients with unrelated, nonmalignant diseases provided the control group for noncarcinoma patients. Finally, some BMAs from unrelated diseases and cytospins of the MCF-7 human breast cancer cell line provided the baseline for background staining.
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Double Immunocytochemical Staining
Double immunostains for NCAM and low molecular weight cytokeratins, as well as for NCAM and leukocyte common antigen (CD45), were carried out in both BMAs from SCLC patients and artificial mixtures (see below) to differentiate, in doubtful cases, true cancer cells from other nonspecifically stained cells (
To co-localize NCAM, cytokeratins, and CD45, we used a protocol combining two APAAP+SABAP staining procedures at RT. For NCAMcytokeratin or NCAMCD45 reactions, the first APAAP+SABAP staining was carried out employing the NCC-LU-246 MAb (1:50) as a primary antibody reagent and New Fuchsin as a chromogen substrate to obtain a red endproduct. After incubation with human serum (5% in TRIS buffer), a second APAAP+SABAP procedure was added using the Cam 5.2 MAb to cytokeratin polypeptide 8 and weakly to 7 (BectonDickinson, Mountain View, CA; dilution 1:10) in combination with the 5D3 MAb to polypeptides 8, 18, and 19 (BioGenex, San Ramon, CA; dilution 1:60) or an anti-human leukocyte common antigen antibody (DAKO-LCA, mixture of clones PD7/26-2B11, Dakopatts; dilution 1:100), respectively, as a second primary antibody reagent and a 0.7% v/v solution of 5% w/v nitroblue tetrazolium (NBT; Sigma) and 2.5% w/v 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma) in Tris buffer as a second substrate to yield a blueblack endproduct (
Artificial Tumor Cell Mixtures for Assessment of Sensitivity
In a set of experiments we used BMAs from healthy volunteers and from patients with unrelated, nonmalignant diseases artificially contaminated with a known number of tumor cells to determine the sensitivity of our method in the detection of NCAM-reactive tumor cells. After sedimentation onto a FicollHipaque gradient density, the cell layer was resuspended at 1 x 106 cells/ml and contaminated logarithmically with the N592 cell line up to 1 tumor cell in 105 mononuclear cells, with additional contaminations at 1:2000, 1:4000, 1:8000, and 1:50,000 cells. Tumor cell dilutions were cytospun to 57.5 x 104 cells/slide, fixed in cold acetone, and immunostained for NCAM with the NCC-LU-246 MAb (1:50) according to the combined APAAP+ SABAP method. Experiments were repeated four to seven times for every dilution and the mean number of immunoreactive tumor cells over 1 x 105 mononuclear cells was recorded.
Evaluation of Immunostained Cells
All estimations of labeled cells, including double stains, tumor cell dilutions, and control group for noncancer patients, were performed independently and blindly by two observers (GP and FP) using an Olympus BH-2 light microscope at a magnification of x400 or x1000 oil immersion. BMAs from SCLC patients were analyzed without knowledge of the patients' identity or stage of disease. For NCAM, cells with thick and continuous membrane staining and morphologically consistent with tumor cells were regarded as immunoreactive. Five cytospins of N592 cell line were examined for each antibody and for each immunostaining method, scoring at least 500 tumor cells in every preparation. A simple semiquantitative grading system for NCAM immunoreactivity was devised: 50% of positive cells +; 5175% of positive cells ++; 76100% of positive cells +++. For BMAs, two to four cytospins were examined for each antibody and for each immunostaining method. Quantitative estimation was thus obtained by counting the number of immunopositive tumor cells over 1 x 105 mononuclear cells. All cytospin preparations were also scanned for intensity of immunostaining (graded subjectively as mild +, moderate ++, or strong +++) and nonspecific background staining (evaluated subjectively as absent -, or present in the form of a granular precipitate of staining +). All scores were assessed by each observer for each antibody and for each immunostaining procedure by counting every case twice. The highest score for each antibody and the highest score between the two observers were thus reported. Positivity for cytokeratins was judged by the occurrence of a strong cytoplasmic staining, whereas positivity for CD45 was identified by a stained ring decorating the cell surface membrane.
Statistical Analysis
The statistical tests used were MannWhitney's test and Fisher's exact t-test. The intra- and interobserver reproducibility was evaluated by analysis of variance and Spearman's rank test, respectively. Survival curves were calculated with the KaplanMeier method (0.05, and all significance levels were of the two-sided type.
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Results |
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N592 Cell Line
NCAM immunostaining results of N592 cell line cytospins are summarized in Table 2 and illustrated in Figure 2. The best results were obtained for both antibodies by using the combined APAAP+SABAP method, which provided the highest number of positive cells and the most intense labeling cell by cell, along with complete absence of nonspecific background staining. In addition, the universal large volume DAKO LSAB Kit AP method strongly stained the large majority of tumor cells with both antibodies, but this was associated with nonspecific granular background staining even after dilution of the reagents. On the other hand, the APAAP and double bridge APAAP techniques, especially with NCC-LU-243, resulted in the staining of fewer cells and in weaker intensity cell by cell, although the nonspecific background staining was very weak.
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Bone Marrow Aspirates
The results of NCAM immunostaining of BMA cytospins are summarized in Table 3. For both antibodies, the combined APAAP+SABAP technique showed a high and comparable number of tumor cells in 24/37 patients (p=NS), strong intensity of immunostaining, and absence of nonspecific background. The DAKO LSAB Kit AP gave almost identical results for the number of stained cases, (23/37), positive cells (p=NS), and the intensity of immunostaining, but it was associated with nonspecific granular background staining. The APAAP method resulted in a smaller number of stained cases (12 to 22/37), with lower numbers of immunoreactive cells (p=NS) and weaker staining intensity. However, nonspecific background staining was absent. The APAAPdouble bridge method stained 19/37 cases with LU-243 and 23/37 cases with LU-246 (p=NS), showing moderate staining intensity and variable occurrence of nonspecific granular background staining. Differences in the prevalence of positive cases (12/37 vs 24/37; p=0.005) and of NCAM-reactive cells (136 vs 710; p=0.019) were found only for the NCC-LU-243 antibody between the APAAP and the APAAP+SABAP techniques, respectively. For all methods of immunostaining, intraobserver reproducibility did not show statistically significant differences in the number of immunostained tumor cells, and a high grade of correlation between the two observers was found (p<0.001; CI 97.0100.0%).
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With all methods of immunostaining, NCAM-positive tumor cells could be seen not only as single cells but also as cell aggregates of variable sizes (Figure 3a and Figure 3b). Both individual and clustered tumor cells were generally larger than normal mononuclear cells. Clusters were formed by two to three to many tumor cells in direct cell-to-cell contact without recognizable intercellular space. The prevalence of clusters and their size increased with the absolute number of tumor cells in BMAs. In fact, 13 patients with a diffuse marrow involvement (more than 70100 positive cells/1 x 105 cells total according to the combined APAAP+ SABAP method) exhibited many clusters, each formed by a variable number of irregularly aggregated tumor cells.
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Control experiments showed that normal bone marrow elements present on cytospin preparations of both clinical material and control group of noncancer patients were completely negative for both NCC-LU-243 and NCC-LU-246, although a few lymphoid cells (Figure 4a) showed a weak and often discontinuous decoration of the membrane, and rare plasma cells (Figure 4b1 and 4b2) and megakaryocytes (Figure 4c) exhibited granular staining of the cytoplasm.
Results of double immunostaining for NCAM and cytokeratins confirmed a definite co-localization of the two markers in the same cells, morphologically consistent with neoplastic epithelial cells (Figure 5a1 and 5a2). Although no cytokeratin+/NCAM- tumor cells were noted, variable numbers of CD45+/NCAM- (Figure 5b, arrow) or CD45+/NCAM+ lymphoid cells (Figure 5c) were also seen. Differential features of NCAM-positive cancer cells vs NCAM-positive non-cancer cells are summarized in Table 4.
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Analysis of Artificial Mixtures
Results of the artificial contamination tests showed that NCAM-positive tumor cells were detected in expected quantities in all mixtures up to the final dilution of 1:105, even if standard deviations were sometimes high (Table 5). Moreover, the intensity of immunostaining of tumor cells was independent of the number of cells detected in every dilution. In doubtful cases, double immunostaining for NCAM and cytokeratins or CD45 was carried out to differentiate true tumor epithelial cells from NCAM-positive noncancer cells. Of note is that the percentage of contaminating cells was one- to 900-fold higher than the percentage of added cells and the enrichment fraction (% of detected cells/% of added cells) was inversely proportional to the percentage of added cells (Figure 6).
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Clinical Implications
Positivity of BMAs evaluated by the combined APAAP+SABAP technique was associated with extensive disease [15/15 (100%) ED vs 10/22 (45.5%) LD] (p<0.001) and positivity of conventional histological examination of marrow biopsy [8/25 (32%) positive BMA vs 0/12 (0%) negative BMA] (p=0.035), whereas no significant relationship was found with age, sex, and performance status. The 22 patients with limited disease presented a significantly lower prevalence of BM involvement than the 15 patients with extensive disease (99 vs 1386 cells, respectively) (p=0.006). The mean amount of marrow contamination in the subgroup of limited disease with positive BMAs was lower than in extensive disease patients (216 vs 1594 cells), although this difference was not statistically significant. The occurrence of BMA involvement significantly affected patients' survival (median survival 9 mo vs 17.5 mo) (p<0.001) (Figure 7a). Other significant indicators were extensive disease (median survival 9 mo vs 16.5 mo) (p<0.001), positivity of conventional bone marrow biopsy (median survival 9 mo vs 15.5 mo) (p<0.001), and performance status lower than 70% (median survival 9 mo vs 16 mo) (p=0.003), whereas age and sex did not affect survival. Combining stage of disease and BMA immunoreactivity at diagnosis, three groups of patients with different prognoses could be identified: Group A, with limited disease and negative BMA (12 patients; median survival 16.5 mo) ("true limited disease"); Group B, with limited disease and positive BMA (10 patients; median survival 13 mo) ("untrue limited disease"); and Group C, with extensive disease and either positive or negative BMA (15 patients; median survival 9 mo). The likelihood of survival was significantly different among groups: A vs B, p=0.017; B vs C, p<0.001; A vs C, p<0.001 (Figure 7b). In multivariate analysis, both extensive disease (hazard ratio = 26.778; CI = 4.380166.009; p<0.001) and limited disease with positive BMA (hazard ratio = 7.399; CI = 1.65533.079; p= 0.003) emerged as independent predictors of a dismal prognosis.
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Discussion |
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Our results can be summarized as follows. First, the choice of the immunostaining technique may influence the detection level of contaminating tumor cells in bone marrow of SCLC patients using anti-NCAM antibodies. Second, BMA contamination by SCLC cells is related to the extension of disease and predicts poor survival. Finally, the presence of tumor cells in BMAs of patients with limited disease appears to identify a subgroup of "untrue limited disease" with reduced life expectancy.
Only the combined APAAP+SABAP method provided for both LU-243 and LU-246 the highest sensitivity, specificity, and staining intensity, with a complete absence of nonspecific background staining on both BMAs and cultured cells. The other three methods tested yielded unsatisfactory results on both materials, with fewer tumor cells revealed, and/or weaker intensity of immunostaining, and/or variable occurrence of disturbing background staining. This observation emphasizes not only the usefulness and the diagnostic value of an immunocytochemical panel approach but also the need for adequate immunostaining techniques for detection of contaminating cells in bone marrow. The higher sensitivity of the combined APAAP+SABAP immunostaining technique is due to the increase in AP molecule numbers brought about at the antigen site through the reaction of the APAAP (two molecules of enzyme for each single molecule of antibody) and biotinstreptavidinAP (a lattice structure containing several enzyme molecules) complexes. Whereas the original Davidoff method combined the APAAP and the avidinbiotinAP complex techniques through a four-step procedure (
To our knowledge, this is the first report in which such an immunostaining amplification system has been evaluated in cytological preparations of BMAs in SCLC patients. Furthermore, we have successfully tested the efficiency of this method in BMAs of gastric and breast cancer patients using several monoclonal antibodies recognizing either epithelial antigens, such as clones MLu-C1, MBr1, MBr8, MOv8, and MOv16 (kindly provided by Dr. Sylvie Menard) and clone BerEp4, or cell cycle-related antigens, such as Ki-67 (unpublished observations). This technique was particularly suitable in cases of false-negative results with conventional immunostaining techniques, such as the APAAP method. In fact, the formation of lattice structures with several enzyme molecules may overcome the presence of small amounts of antigen. The reliability of this amplification system was assessed not only by the morphological recognition of immunoreactive cells as tumor cells but also by the co-localization of NCAM and cytokeratins. Moreover, the complete absence of nonspecific background staining confirmed the high specificity of the method. Crossreaction with plasma cells and megakaryocytes was easily recognizable as nonspecific because it was confined to the cytoplasm, persisted in the negative control experiments using nonrelated IgG, and the cell morphology was clearly not consistent with that of epithelial cells. We believe that the morphology should be the basic principle of immunocytochemical detection of isolated epithelial cells in bone marrow because a potential contribution to nonspecific staining has been reported by normal mononuclear cells directly reactive to AP (
The high sensitivity of the combined APAAP+ SABAP method enable us to identify tumor cells in expected amounts in all dilution tests, even though the number of cells detected could be higher than that of added cells (Table 5). This can certainly be explained by the varying distribution of tumor cells during centrifugation within cytospin preparations, especially in cases with low NCAM-positive tumor cell counts, probably due to the different density of cancer cells compared with that of marrow cells (
Few studies have been aimed at increasing the sensitivity of AP complex-based signal systems for immunocytochemistry. These approaches included repeated applications of signal complexes (
Regarding clinical implications, our data, although preliminary, show that BMA contamination by tumor cells is predictive of poorer survival (p<0.001) (Figure 7). However, this phenomenon might reflect the very poor prognosis of patients with extensive disease, because in our series positive BMAs were more common in patients with extensive disease (p<0.001). Consistently, we detected at least one order of magnitude more tumor cells in BMAs of patients with extensive disease (1386 cells) than in those with conventionally assessed limited disease (99 cells) (p=0.006). This observation also confirms previous reports showing that the persistence of residual marrow disease at clinical remission (
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Acknowledgments |
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Supported by grants from the Ministry of University and Scientific Research (60%), Rome, Italy.
Received for publication March 26, 1999; accepted March 30, 1999.
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