Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme-linked immunosorbent assay.

BACKGROUND: Point mutations of the ras protooncogene, primarily within codons 12 and 13, are commonly identified in colorectal carcinomas and large adenomas. Despite data suggesting that ras genotyping may have clinical significance with respect to colorectal cancer screening and prognosis, more widespread use has been limited because of the lack of a suitable assay system. The principal objective of this study was to assess the feasibility and validity of a qualitative enzyme-linked immunosorbent assay (ELISA) for detecting the four most common ras mutations in human colorectal tumors at the protein (p21ras) level. METHODS: Tissue homogenates (11-121 micrograms) from endoscopically or surgically resected colorectal adenomas, carcinomas, and normal mucosae were evaluated by a commercially available ELISA (Oncogene Science, Inc. Cambridge, MA) for mutant p21ras containing arginine position 12 (arg12), valine position 12 (val12), aspartate position 12 (asp12), and aspartate position 13 (asp13) amino acid substitutions. Portions of the same tissue from an initial series of 27 specimens also were subjected to mutant-enriched polymerase chain reaction (PCR) and/or PCR amplification with subsequent DNA sequence analysis to validate the ELISA data. RESULTS: Forty-seven adenomas, 9 carcinomas, and 14 normal mucosae were assayed. Mutations were identified in 16 (34%) of the adenomas (7-asp12, 7-val12, 2-asp13), 3 (33%) of the carcinomas (1-asp12, 1-arg12, 1-asp13), and none of the normal mucosae by ELISA: Polymerase Chain Reaction and DNA sequencing analyses demonstrated identical results for 21 of the 23 (91%) and 14 of 16 (88%) homogenates tested, respectively. The ELISA demonstrated an overall sensitivity of 80-86%, specificity of 90-92%, positive predictive value of 86-100%, and negative predictive value of 86-91%. CONCLUSIONS: The ELISA is a feasible and valid approach for identifying p21ras mutations in human colorectal adenomas and carcinomas.

A three-dimensional system for long-term culture of human colorectal adenomas.

Studies of the adenoma-carcinoma sequence in the colon and rectum have been limited by the paucity of experimental models of adenoma growth and progression. Progress recently was reported in the development of monolayer culture systems. The principal objective of this study was to develop a primary culture system for colorectal adenomas that would simulate three-dimensional in vivo growth. We used a calcium alginate encapsulation technique that was previously described for established tumor cell lines. Briefly, fresh resected specimens were washed, minced into small multicellular particles called microadenomas, and encapsulated in 1% calcium alginate pellets. The pellets were maintained in minimum essential medium containing 10% fetal bovine serum at 37 degrees C in humidified atmosphere of 95% air, 5% CO2. Ten of eleven adenomas, including six tubular, three tubulovillous, and one villous have been successfully cultured for 34 to 162 days. Cell viability was confirmed histologically by light and electron microscopy. The cells were characterized as epithelial by morphologic features and ultrastructural studies, which showed a high degree of cellular differentiation, including villous brush borders and many desmosomes. Both tubular and villuslike structures have been observed in vitro, correlating in some cases with the histology of the parent adenoma. Measurements of proliferative activity by [3H]thymidine autoradiography or immunohistochemical staining with the monoclonal antibody Ki-67 demonstrated growth fractions of 9% to 25%. A simple, highly efficient primary culture system was developed for the long-term maintenance of adenomas that promotes three-dimensional growth patterns and growth rates analogous to those seen in vivo. This model provides an opportunity to develop an experimental system for longitudinal studies of pathologic and molecular parameters in adenoma progression to carcinoma.

Characteristics of invasive and noninvasive human colorectal adenocarcinoma cells.

A human colon adenocarcinoma cell line (LoVo) established from a lymph node metastasis was used to study properties associated with invasive tumor cells. Human amniotic membranes were used as invasion barriers to select highly invasive and noninvasive subpopulations of cells from the parent LoVo line. Enriched subpopulations were compared with respect to parameters associated with invasion. The invasive cells were more invasive in vitro and more highly sialylated than either the parental or noninvasive line. Invasive cells migrated more strongly in vitro toward gradients of soluble and insoluble laminin, and their augmented migration correlated with increased adhesion and spreading on laminin-coated substrata. Invasive cells also had the highest level of specific laminin-binding activity. Thus, the invasive cells were shown to possess specific properties that correlated with their successful traversal of the human amnion membrane.

Chemotherapy screening assay using 3-dimensional cell culture.

A new alginate culture method (ACM) was used to test the effects of vincristine and 5-fluorouracil (5-FU) on the commonly used human colon carcinoma cell line HT-29. Colorimetric analysis of viability following treatment with physiologically tolerated levels of vincristine showed significant reduction of the surviving cell population. Hematoxylin and eosin staining of sections of treated organoid growths corroborated the colorimetric analyses. The ACM is inexpensive, rapid and extremely versatile. Many alginate beads containing "mini-tissue" growths of many cell lines can be used simultaneously to evaluate numerous chemotherapeutic agents in an "in vivo-like" environment.

L-azaserine induced preneoplasia in the rat pancreas. A morphometric study of dietary manipulation (lipotrope deficiency) and ultrastructural differentiation.

Putatively preneoplastic, pancreatic atypical acinar cell foci (AACF) and nodules (AACN), collectively termed atypical acinar cell lesions (AACL), were induced in male Lewis rats by L-azaserine (300 mg/kg body weight [bw] in divided doses). Rats given carcinogen and then fed a lipotrope deficient (LD) diet developed a significantly greater number of larger lesions than animals fed complete diet throughout the experiment. It is suggested that lipotrope deficiency plays a promoting role in this model of pancreatocarcinogenesis. Ultrastructural morphometric studies of AACF, when compared to control tissues, revealed the following significant results: 1) a decrease in surface area of cell cytoplasm with no change in nuclear area, and hence increased nucleus/cytoplasm (N/C) ratio; 2) a reduction in size and uniformity of zymogen granules; and 3) an increase in number of granules per microns 2 of cell. The results suggest that arrested development of the AACF cells is associated with reduced cytoplasm and zymogen production per cell. AACL may be eosinophilic due to an overall increased concentration of zymogen in these hyperplastic lesions and not because individual acinar cells in the AACL contain an increased amount of zymogen or are "zymogen-rich," as has been reported.

Laminin expression by human pancreatic carcinoma cells in the nude mouse and in culture.

The abilities of 4 established human pancreatic tumor cell lines (PANC-1, CAPAN-1, AsPc-1, and BxPc-3) to synthesize and to maintain laminin-containing basement membranes when grown in the nude mouse have been examined and compared with synthesis of the glycoprotein laminin by these tumor cells when grown in culture. Immunohistochemical visualization of basement membrane integrity within the transplanted tumors employed a monoclonal antibody specific for human laminin to clearly distinguish between human tumor-produced laminin and murine basement membranes. This technique demonstrated strikingly different degrees of basement membrane formation and laminin distribution for the 4 biologically diverse pancreatic tumors. Basement membranes were present within the differentiated, less invasive tumors, whereas structure basement membranes were absent in the poorly differentiated, invasive tumors. Regardless of their propensity to produce basement membranes in vivo, all 4 pancreatic tumor cell lines continued to synthesize laminin in culture, as was determined by immunologic assays. The most invasive cell line, AsPc-1, released large quantities of soluble laminin into conditioned culture medium. The less invasive, well-differentiated tumor cells did not release laminin into the medium, but they retained the laminin produced by them within the cell layer. The combination of in vivo and in vitro studies indicates that the extent of basement membrane loss by these human pancreatic tumors is not due to an inherent inability of the tumor cells to synthesize the structural component laminin.

Ultrastructural differentiation and CEA expression of butyrate-treated human pancreatic carcinoma cells.

The effects of butyrate (a biological response modifier) on cellular morphologic features and carcinoembryonic antigen (CEA) expression of human pancreatic carcinoma cells were studied and compared in a well-differentiated, CEA-producing cell line (CAPAN-1), and a poorly differentiated cell line (PANC-1). Butyrate treatment resulted in the acquisition of phenotypic traits commonly attributed to increased "differentiation," including a twofold increase in doubling time, decreased saturation densities, and approximately 50% reduction in colony forming efficiency in both cell lines. Elongation and flattening of cells with extending cellular processes were seen by light microscopy. Significant ultrastructural changes were seen only in the PANC-1 cells, including an increased number of intercellular desmosomes, tonofilaments, and lipid droplets. In contrast, to the coarsely clumped nuclear chromatin (heterochromatin) of untreated PANC-1 cells, the nuclei of the butyrate-treated cells consisted of finely dispersed chromatin (euchromatin). CAPAN-1 cells responded to butyrate with increased CEA synthesis and release. This effect was greatest in the stationary growth phase. Butyrate had no effect on the already low rate of CEA synthesis by PANC-1 cells. These studies suggest that CEA synthesis and state of differentiation are affected independently by butyrate treatment and that the original tumor phenotype plays an important role in response to such treatment.

Diversity of adhesion to basement membrane components of human pancreatic adenocarcinomas.

Human carcinomas of the pancreas are aggressive tumors which traverse basement membrane barriers during invasion and metastasis. In order to examine the relationship of pancreatic tumor cells to basement membranes, we analyzed and compared the capabilities of four biologically different human pancreatic adenocarcinoma lines to adhere to substrate coated with purified basement membrane constituents. Each of the four cell lines adhered readily to purified laminin in a dose-dependent manner, although differences were noted in the time required for optimum attachment. Significant variations in the abilities of the cell lines to attach to purified fibronectin were evident both in concentration dependence and in the time required for attachment and spreading. Adhesion to type IV collagen was negligible for two of the four tumor lines but addition of soluble laminin or fibronectin augmented attachment. The other two cell lines attached only moderately to type IV collagen and this attachment was not enhanced by soluble laminin or fibronectin. When laminin or fibronectin was coated directly over type IV collagen, attachment of all four cell lines was comparable to that for the glycoproteins alone. Although the tumor lines were all established from human neoplasms of similar histological origin and retained the ability to adhere to intact basement membranes prepared from human amnion, they exhibited various patterns of attachment to laminin, fibronectin, and type IV collagen.

A new method for the three-dimensional in vitro growth of human cancer cells.

A method has been developed which allows, for the first time, the in vitro growth of human tumor cells in calcium alginate gel microcapsules. The three-dimensional matrix is permeable to macromolecules of at least 200 000 molecular weight (MW) (e.g., carcinoembryonic antigen) and provides an environment for organized growth and differentiation more comparable to in vivo growth than monolayer culture. This inexpensive, nondestructive procedure allows studies of morphological, biochemical and immunochemical parameters of human tumor cells within a single model.

Monoclonal antibodies to carcinoembryonic antigen produced by somatic cell fusion.

Hybridoma cell lines secreting monoclonal antibodies to carcinoembryonic antigen (CEA) were generated by fusing mouse immune lymphocytes with the mouse myeloma variant cell line, NS-1. Antibody secreted by one cloned hybrid cell line could bind only a select portion of the CEA bound by the commercially available goat anti-CEA antiserum used in clinical assays. Radiolabeled CEA could be purified on a monoclonal antibody affinity column. Incorporation of this purified radiolabeled CEA in a double-antibody solid-phase assay with goat anti-CEA antiserum led to an approximately 2.5-fold increase in sensitivity of the assay. Genetically stable hybrid clones may be sources of virtually unlimited quantities of such antibodies which may have potential utility in improving the cancer specificity of clinical assays.

Carcinoembryonic antigen gradients between plasma and malignant ascites: use in detecting peritoneal and liver metastases.

In the study of 19 patients with malignant ascites and elevated plasma carcinoembryonic antigen (CEA) (greater than 2.5 ng/ml) and/or ascites CEA (greater than 10 ng/ml), two patients emerged: 1) The 10 patients with higher ascitic fluid than plasma CEA levels (medians: 230 and 10.6 ng/ml, respectively) had exudates and intraperitoneal cancer but usually had no hepatic metastases. 2) The 9 patients with lower ascitic fluid than plasma CEA levels (medians: 29 and 140 ng/ml, respectively) had transudates and negative cytology examinations but did have demonstrable liver metastases. Determination of ascitic fluid and plasma CEA gradients in patients with malignant ascites may help localize, with potentially therapeutic importance, the site of metastases.

The disparity of indirect and direct zirconyl-gel assays for carcinoembryonic antigen.

Experience with the zirconyl phosphate gel (Z-gel) radioimmunoassays for plasma CEA levels below 20 ng/ml (the indirect method) and for levels greater than 20 ng/ml (the direct method) has shown that a disparity of values exists, caused by shifting from one assay to the other. This disparity is at least partially due to PCA-labile proteins reacting in the direct assay. It may be constant for individual patients but varies among patients. The magnitude of this disparity is independent of the CEA level (above 15 ng/ml).

Carcinoembryonic antigen assay of ascites and detection of malignancy.

Levels of carcinoembryonic antigen (CEA) activity were measured in 105 unselected samples of ascitic fluid submitted for routine cytologic analysis to ascertain whether this assay was useful in the detection of malignancy. The highest CEA level found in the 70 specimens of "benign" ascites was 10 ng/ml. Using values greater than 10 ng/ml as indicating a cancerous effusion, CEA assay successfully detected 14 of the 29 malignant ascites studied. Cytology, on the other hand, detected only 12 of these fluids. Combining the two methods increased the yield to 20. The CEA assay alone thus detected more than one fourth of these malignant fluids. The assay was particularly useful in detecting malignant transudates, nearly half of which had elevated CEA levels despite negative cytologic findings. The CEA assay of ascites thus showed promise as an adjunct to cytology in the detection of malignant ascites when used as part of a complete clinical and laboratory assessment.

Carcinoembryonic antigen levels in benign and malignant pleural effusions.

One hundred ninety-one unselected fluid specimens submitted routinely for cytologic examination were assayed to determine whether the measurement of carcinoembryonic antigen (CEA) levels in pleural effusions is useful in detecting malignancy. The mean +/- SD CEA level of 103 benign effusions was 4.1 +/- 2.9 ng/ml. Only one benign effusion had a level greater than 12 ng/ml (18 ng/ml). Benign inflammatory effusions (pneumonia, empyema) had a higher mean CEA activity (6.2 +/- 3.4) than effusions caused by congestive heart failure (2.9 +/- 1.5) (p less than 0.001). Twenty-four (34%) of 70 malignant effusions had a CEA level greater than 12 ng/ml, and 28 (40%) were "positive" by cytologic study. Thirty-eight (54%) were detected by one or both methods. Ten malignant effusions were positive by CEA (greater than 12 ng/ml) alone. These data suggest that the determination of CEA activity levels, when used in conjunction with other clinical findings, may be useful in detecting malignant pleural effusions.

Demonstration and immunochemical characterization of carcinoembryonic antigen in human pancreatic juice.

Pancreatic juice collected from 10 patients without evidence of malignant disease of the pancreas or other organs was pooled, extracted, and fractionated by Sepharose 6-B and Sephadex G-200 gel filtration. The carcinoembryonic antigen (CEA) activity in the material was demonstrated and studied by: a) radioimmunoassay, b) competitive binding to antibodies against CEA, c) precipitin inhibition, and d) Ouchterlony analysis. The immunochemical identity of the active material to CEA purified from liver metastases of colon cancer was demonstrated.