Acridine orange flow cytometric analysis of renal cell carcinoma. Clinicopathologic implications of RNA content.

To explore the potential role that ribonucleic acid (RNA) content analysis may have in the assessment of primary renal cell carcinomas (RCC), biparametric flow cytometric (acridine orange) measurements for DNA/RNA were obtained on 108 fresh neoplastic specimens. RNA content was divided into low and high groups, based on the average RNA content in normal kidney controls. High RNA content was significantly correlated with aneuploidy, high proliferative index, high nuclear grade, cytoplasmic granularity, and large tumor size. No correlation was found between RNA content and patients' sex, race, and clinical stage of the carcinomas. When diploid RCCs were separately analyzed, high RNA content was correlated with high nuclear grade, large tumor size, high clinical stage, and cytoplasmic granularity. There was no correlation between RNA content and the patient's sex or race or the neoplasm's proliferative index. Of the 16 patients that relapsed (5 diploid and 11 aneuploid), four of the diploid and all 11 aneuploid neoplasms displayed high RNA content. The authors' data show that RNA may be a valuable objective and quantitative parameter in the clinicopathologic assessment of RCC.

Modulation of pro-epidermal growth factor, pro-transforming growth factor alpha and epidermal growth factor receptor gene expression in human renal carcinomas.

We have analyzed the expression of the genes for the precursors of epidermal growth factor (pro-EGF) and transforming growth factor alpha (proTGF-alpha) as well as for the EGF receptor in tissue specimens of a large number of adult patients with renal cell carcinoma. Since normal kidney tissue was available from the same patients we could directly compare the expression of these genes in tumors with that in adjacent normal renal tissue. Our experiments reveal underexpression of the proEGF gene in all tumors analyzed (21 of 21) and overexpression of the genes for proTGF-alpha (33 of 33 analyzed) and EGF receptor (22 of 23 analyzed) in tumor samples, when compared with normal kidney tissue. The expression of the proTGF-alpha gene appeared to depend on grade and differentiation of the tumor, since well differentiated tumors (grade 1) expressed more proTGF-alpha mRNA than the adjacent normal tissue but significantly less than poorly differentiated tumors (grade 2 or 3), which are the most aggressive ones. In none of these tissue specimens did we find, by Southern analysis, amplification of the proTGF-alpha or EGF receptor gene. Therefore, overexpression of these genes must be due to another effect, perhaps an alteration of their mRNA turnover. Although the EGF receptor gene (c-erbB1) is overexpressed in nearly all carcinomas analyzed, there was no linear coexpression with the proTGF-alpha gene. In contrast, transcription of the proEGF gene was completely turned off in tumor tissue. Although we have found by restriction fragment length polymorphism analysis, in one of three tumor samples, evidence for a somatic mutation within the proEGF gene, we do not know yet, due to the limited number of Southern analyses, whether this somatic mutation is causally involved in the decrease of proEGF mRNA expression and, hence, is representative of renal cell carcinoma. To our knowledge, this is the first observation on primary tumor tissue in humans that upon malignant transformation the gene for a polypeptide growth factor gene is underexpressed.

Cell differentiation in Wilms' tumor (nephroblastoma): an immunohistochemical study.

Using an indirect immunoperoxidase technique, we tested frozen specimens from 12 Wilms' tumors with monoclonal antibodies (MoAbs) reacting against a large panel of molecules including laminin, fibronectin, cytokeratin, vimentin, villin, CD24, CALLA/CD10, CR1, CD26, class I and class II major histocompatibility complex (MHC) molecules, and endothelium factor VIII. These molecules were chosen because they are markers of specific segments of the mature kidney and because their loss or acquisition is indicative of different steps of human nephrogenesis. KI67 MoAb was used to evaluate the proliferating activity of the cells. The blastemal component (cell compact areas) of Wilms' tumors consisted of vimentin-positive cells with a fibronectin network. However, signs of epithelial maturation were present in compact areas where cytokeratin-positive cells producing laminin were observed. The cells exhibited a high degree of proliferating activity. The tubule formations consisted of cytokeratin-positive cells and had a defined laminin border. All the cells, whether in compact areas or in tubules, were strongly CD24-positive. Some tubular formations showed signs of proximal maturation with the presence of CALLA, CD26, and even villin. In four cases class I-MHC molecules were expressed by some tubular cells. Large cystic cavities present in five cases were edged by cytokeratin, CD24-positive cells, or by vimentin, CALLA, CR1-positive cells. Some glomeruloid bodies, present in two cases, were also composed of vimentin, CALLA, and CR1-positive cells which correspond to the mature podocyte phenotype. The interstitial tissue contained mainly laminin and fibronectin network with macrophages and few CD3 lymphocytes. The presence of large cells with muscular differentiation was noted; round vimentin and CD26-positive cells were also seen. The endothelial cells of the vessels exhibited vimentin, factor VIII, and class I and class II MHC molecules as do mature cells, but in some cases the endothelial cells lacked class II molecule expression and were CALLA-positive. These results which confirmed and extended those previously described show that cell differentiation in Wilms' tumor mimics that observed during metanephros development. Moreover, this study shows that tumoral cells in nephroblastoma share several antigens with cells from lymphoid lineage (CD24, CALLA, and CD26) as do developing and mature kidney cells. Such cell phenotype dissection provides a useful and reliable tool for testing the influence of various factors on the development of hetero-transplanted or cultured Wilms' tumors.

[Tumor heterogeneity of renal cell carcinoma as analyzed by flow cytometry].

DNA content in renal cell carcinoma was investigated to examine the tumor heterogeneity and was correlated with their morphologic grades. A total number of 147 paraffin-embedded samples (2-6 samples with a mean of 5) from 30 tumors were analyzed by flow cytometry. DNA aneuploid patterns were demonstrated in 5 of 9 grade 1 tumors (56%), 15 of 18 grade 2 tumors (83%) and 3 of all grade 3 tumors (100%), while aneuploid DNA histograms were exhibited in 20 of 57 grade 1 samples (35%), 45 of 81 grade 2 samples (56%) and 8 of 9 grade 3 samples (89%). DNA aneuploid patterns were demonstrated more significantly in grade 2 and 3 samples than in grade 1 samples (p less than 0.02 and p less than 0.01, respectively). Consequently, 73 samples (50%) showed DNA aneuploid patterns and 74 samples exhibited DNA diploid patterns. Eleven tumors (37%) showed homogeneous DNA ploidy patterns (7 tumors were diploid and 4 tumors were aneuploid only), while 19 tumors (63%) showed DNA heterogeneity, 17 of these 19 tumors demonstrating diploid as well as aneuploid samples. DNA heterogeneity was not found when the tumor samples examined were fewer than 3 samples. However, 19 of 25 tumors (76%) in which more than 4 samples were examined showed DNA heterogeneity, the incidence of which tended to be increased with upgrading of the tumor--3 of 9 (33%) in grade 1 tumors, 13 of 18 (72%) in grade 2 tumors and 3 of all (100%) in grade 3 tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of polysialic acid and the neural cell adhesion molecule N-CAM in Wilms tumor and their subcellular distributions.

In previous studies we have reported that polysialic acid is an oncodevelopmental antigen in human kidney but its relationship to the neural cell adhesion molecule (N-CAM) remained undefined. In the present study, we showed by the combination of immunoprecipitation and immunoblotting that renal polysialic acid is a structural component of N-CAM polypeptide and that two highly sialylated N-CAM isoforms of approximately 120 kDa and 140 kDa existed in Wilms tumor. The presence of a cell surface coat composed of polysialic acid and N-CAM was revealed by immunoelectron microscopy, and morphological evidence for its involvement in modulating cell-cell adhesion has been provided. Furthermore, highly sialylated N-CAM was detectable extracellularly. N-CAM immunolabeling was present in compartments from the nuclear envelope to the plasma membrane. However, polysialic acid was only detectable at the cell surface suggesting that in Wilms tumor cells sialyl polymer synthesis may occur partially or exclusively at this site.

A prospective comparison of DNA quantitation by image and flow cytometry.

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

Heterogeneity of polyoma tumors in hamsters: analysis of cytoskeletal proteins and viral gene expression.

Murine polyomavirus-induced hamster tumors revealed an unexpected heterogeneity with respect to patterns of cytoskeletal proteins expressed in different visceral and subcutaneous tumors and with respect to viral gene expression early during tumor outgrowth. All tumors analyzed expressed vimentin. Desmin was found in all heart tumors, to variable degrees in kidney tumors and in trace amounts only in 1 out of 4 s.c. tumors. The alpha-smooth-muscle actin isoform was observed in heart tumors only and was restricted to structures that we interpret as being proliferating pericytes or proliferating smooth-muscle cells of the media. In kidneys of infected newborn animals and before the appearance of macroscopic tumors, viral early mRNAs were transcribed from free viral genomes. In tumor tissue the size of the viral transcripts was altered, suggesting that they were transcribed from integrated viral DNA. Since in each tumor discrete bands of viral RNAs were detected, individual tumors arose presumably from single cells and one functional integration event. In contrast, in situ hybridizations of kidney tissue and tumors showed large quantitative differences in viral gene expression not only between different tumors but also between individual cells of the same tumor.

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Immunohistochemical demonstration of epidermal growth factor in human gastric cancer xenografts of nude mice.

Thirty-two surgical specimens and three cell lines of human gastric cancers were used for subcutaneous transplantation into nude mice, resulting in the establishment of eight (25%) xenografts from the surgical specimens and two (67%) from the cell lines. The localization of epidermal growth factor (EGF) in the surgical specimens and cell lines of the gastric cancers and their xenografts in nude mice was then investigated immunohistochemically. Epidermal growth factor was stained in the cytoplasm of the cancer cells, being detected in 16 (50%) of the 32 surgical specimens and in all of the cell lines. Seven (44%) of the sixteen EGF-positive surgical specimens and one (6%) of the 16 EGF-negative ones were tumorigenic in nude mice. All of the xenografts in nude mice were positive for EGF. The tumorigenicity of human gastric cancer xenografts in nude mice may, therefore, be correlated with the presence of EGF in cancer cells.

[Flow cytometric analysis of renal cell carcinoma].

Forty cases of renal cell carcinoma were studied retrospectively by flow cytometry and DNA contents of the cancer cells were measured. The results indicated that the incidence of aneuploid tumor was 57.5% (23/40), diploid tumor or quasi-diploid tumor 42.5% (17/40). DNA ploidy was strictly correlated to histopathological grade, clinical stage, cancer cell type and survival time. Therefore, analysis of cellular DNA ploidy of renal cell carcinoma is of prognostic value for renal cell carcinoma.

Tumour DNA content and skeletal metastases in renal cell carcinoma. A preliminary report.

The DNA content of the tumour cells in 10 patients with primary renal cell carcinomas was analysed; from six of the patients skeletal metastases were also studied. Four patients had homogenously diploid primary tumours, with solitary metastases. Six patients had aneuploid primary tumours, three with solitary and three with multiple metastases. In two patients radical excision of diploid metastases resulted in long disease-free intervals. Patients with diploid tumours survived significantly longer than patients with aneuploid tumours. These results indicate that tumour DNA content might be a useful prognostic indicator. The measurement of DNA content may be a suitable method of identifying those patients likely to survive long enough to benefit from major surgical resection and reconstruction.

[The capacity of glucocorticoid receptors for activation as a possible criterion of the hormonal sensitivity of human tumors]. / Soposobnost' retseptorov gliukokortikoidov k aktivatsii kak vozmozhnyi kriterii gormonal'noi chuvstvitel'nosti opukholei cheloveka.

For the functional activity evaluation of glucocorticoid receptors (GR) a specific method for their activated form determination was developed on the experimental model of the rat liver. The method is based on the interaction of GR with DNA-cellulose. It has been shown that 26-41% of the total rat liver GR are able of undergoing activation and being bound to DNA-cellulose. Activated GR were studied in 88 renal tumours and in 37 melanomas. 20% of renal tumours and 33% of melanomas contain GR which are not able to undergo activation. The number of activated GR in most cases is much lower than the total GR number. There is no direct correlation between the concentrations of total and activated GR, as well as between the total GR content and the percent of activated forms in the whole receptor pool. It appears that the study of activated GR may give more reliable information about the hormone sensitivity of tumours than the determination of the total receptor content.

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats.

Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any well-known organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.

Immunoelectron microscopic investigation of surface coat of Wilms tumor cells. Dense lamina is composed of highly sialylated neural cell adhesion molecule.

In previous studies we demonstrated the presence of polysialic acid in Wilms tumor by immunohistochemistry and immunoblotting. We now show by immunoelectron microscopy that the cell surface coat of Wilms tumor cells consists of a layer of amorphous material containing polysialic acid but not detectable amounts of laminin, laminin-nidogen complex, or low density proteoglycan. Therefore, Wilms tumor cells are covered by a highly developed and chemically specialized cell surface coat that does not represent a basement membrane, although it bears some structural similarities. Polysialic acid is present on neural cell adhesion molecule that exists in Wilms tumor as two isoforms of approximately 120 and 140 kDa. The cell surface coat exhibits variation in its thickness along the plasma membrane of a single tumor cell, and the variation is inversely related to the extent of cell-cell contact. It is therefore proposed that polysialic acid may modulate the behavior and invasive potential of Wilms tumor cells.

Detection of drug resistance and P-glycoprotein in human renal cell carcinomas.

Expression of the multidrug-resistance gene product P-glycoprotein (P-170) was screened in 21 untreated human renal cell carcinomas using monoclonal antibodies (265/F4, C219) and immunoperoxidase staining. The inherent drug resistance of the same samples against doxorubicin was established by a short-term chemoresistance test in order to investigate the association between the expression of P-170 and intrinsic drug resistance in kidney cancers. P-glycoprotein could be demonstrated in 10 cases. Using the short-term test for predicting resistance, 17 resistant and 4 sensitive cancers were found. In vitro 10 of 17 resistant tumors revealed an increase of P-glycoprotein. On the other hand, in the sensitive tumor in vitro, an expression of P-glycoprotein could not be demonstrated. This investigation reveals that intrinsic drug resistance exists in many renal cell carcinomas and it is associated at least in part with increased expression of P-glycoprotein. The immunohistochemical results suggest that the presence of the P-glycoprotein may be useful as a marker for screening the multidrug-resistant phenotype in renal cell carcinomas and as an indicator of the therapeutic efficacy of multidrug-resistant kidney cancers.

Expression of transforming growth factor alpha in normal human adult kidney and enhanced expression of transforming growth factors alpha and beta 1 in renal cell carcinoma.

Normal kidney and renal cell carcinoma tissues from ten patients were studied for mRNA and DNA for both transforming growth factors alpha and beta 1. Northern and Southern hybridizations were conducted on samples extracted from the solid tumor and surrounding normal tissues and two tumor-derived cell lines. Low levels of constitutive expression of TGF-alpha mRNA were detected in all normal kidney tissues; six of the ten patients, however, demonstrated an increased (2- to 8-fold) expression of TGF-alpha in the tumor versus normal kidney as determined by densitometry of RNA blots. All ten patients had elevated mRNA levels for TGF-beta 1 in the tumor (2.5-to 22-fold increase) relative to normal kidney. Two tumor-derived cell lines also expressed TGF-alpha and TGF-beta 1 mRNA. Southern blot hybridization of the DNA extracted from the normal tumor pairs revealed no gene amplification or gross rearrangement for either the TGF-alpha or TGF-beta 1 genes. These results demonstrate the expected constitutive expression of TGF-beta 1 by normal kidney; however, the constitutive expression of TGF-alpha by Northern blot analysis in normal adult human kidney is previously unreported. Enhanced expression of TGF-alpha and TGF-beta 1 mRNA in solid tumor may be related to the development of renal cell carcinoma.

[The study of plasminogen activator in renal cell carcinoma with special remarks on urokinase type plasminogen activator].

Plasminogen activators (PA) have been reported to be associated with fibrinolysis. The amounts of PA in urine, plasma, and tissues of patients with renal cell carcinoma were determined by measuring the amounts of two kinds of antigens, urokinase type (u-PA) antigen and tissue type (t-PA) antigen, by highly sensitive enzyme-immunoassay. The u-PA antigen level in urine showed neither daily variation nor age-relationship. It was, however, significantly higher (164.2 +/- 93.5 x 10(2) U/gCr) in patients with renal cell carcinoma than in healthy subjects (56.8 +/- 22.4 x 10(2) U/gCr) (p less than 0.01). The amount of u-PA antigen in urine tended to be higher in patients with high grade or stage cancer than in those with cancer of low grade or stage, though not statistically significant. The u-PA antigen content in tissues appeared elevated in tumors (8.90 +/- 6.00 x 10(-1) U/g wet tissue) in comparison to normal renal cortex and medulla. However, the difference was not significant, as the cancer samples consisted of various tissue components including necrotic or hemorrhagic tissue in addition to cancer cells. Although the t-PA antigen content in urine was too immeasurably small in 29% cases by the present method, there was no significant difference between patients with renal cell carcinoma and healthy subjects. The plasma level of t-PA antigen tended to be elevated in renal cell carcinoma group (7.87 +/- 5.60 U/ml) compared to the control group (5.7 +/- 2.19 U/ml), but no significant difference was present between them.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of the 1 alpha, 25-dihydroxyvitamin D3 receptor concentration and the DNA content in renal cell carcinomas.

A role has been suggested for 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in cell proliferation and differentiation. Therefore the concentration of the 1,25-(OH)2D3 receptors and DNA-ploidy was measured in 22 renal cell carcinomas. No relation was found, the mean 1,25-(OH)2D3 receptor concentration being 8.4 fmol/mg of protein (range 2.8-15.9) in diploid tumors and 7.0 fmol/mg of protein (range 0-27.8) in DNA aneuploid tumors. The aneuploid tumors (11 out of 22) had a heterogeneous DNA content in 9 out of 11 cases. At the time of operation, no patient had metastases. In this prospective study, one out of 9 patients with DNA aneuploid tumor had died of renal cell carcinoma (observed 33-58 months, median 42 months). None of 10 patients with diploid tumors had died (observed 40-56 months, median 52 months).

Epidermal growth factor receptor gene expression in normal human kidney and renal cell carcinoma.

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein detected on many cells and tissues including neoplastic and normal kidney. EGFr binds the mitogenic polypeptide hormone epidermal growth factor (EGF) as well as EGF-related transforming growth factor-alpha (TGF-alpha). Increases in EGFr gene expression and protein production have been implicated in the development of the malignant phenotype for certain cancers. To determine if alterations in EGFr gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from ten patients with advanced renal cell carcinoma were analyzed for EGFr mRNA content by Northern blot hybridization. The EGFr gene was constitutively expressed in 90% of normal kidney samples. In seven out of nine evaluable patients, tumors expressed 1.7 to 8.4 times more EGFr mRNA than corresponding normal tissue. Two patients showed no elevation of tumor EGFr mRNA and one patient had no identifiable EGFr transcripts in either neoplastic or normal kidney. Expression of EGFr mRNA was also detected in three tumor-derived and two established renal cell carcinoma cell lines. EGFr transcripts were not found in tumor infiltrating lymphocytes (TIL). These findings suggest that overexpression of EGFr mRNA may be associated with malignant transformation in renal cell carcinomas.