Induction of cells with phenotypic features of neuronal cells by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate in a human parotid gland adenocarcinoma cell line in culture.

A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Lesiones epiteliales ductales y adenocarcinoma pancreático / Ductal epithelial lesions and pancratic carcinoma

Epithelial alterations in pancreatic ducts may have a premalignant nature. We compared the incidence of different lesions in 49 pancreatic cancer specimens comapred to 100 controls. Simple hyperplasia, mucosecretory metaplasia and pyloric metaplasia were evenly distributed among groups. In contrast, papillary hyperplasia, intestinal metaplasia and dysplasia were significantly more frequent in pancreas with carcinoma. Therefore, these lesions may have a precancerous nature

Independent prognostic value of ploidy in colorectal cancer. A prospective study using image cytometry.

In a prospective study, the DNA content of Feulgen-stained nuclei obtained from fresh samples of 211 colorectal adenocarcinomas was evaluated by means of image analysis. The DNA histogram classification took into account aneuploidy and S-phase fraction for diploid cases. No significant relationship was found between ploidy and sex, age, preoperative carcinoembryonic antigen (CEA), size of the tumor, histologic differentiation, or Dukes' stage. Aneuploidy was more frequently encountered in distal tumors. Preoperative CEA, histologic differentiation, Dukes' stage, and ploidy were individually associated with overall survival. In Dukes' A, B, and C tumors, patients with normal and elevated CEA had no significant difference in overall survival. A relationship was apparent between disease-free survival and site, histologic differentiation, Dukes' stage, and ploidy. Multivariate overall survival analysis did not reveal independent prognostic significance of ploidy when all Dukes' stages were considered. In contrast, Dukes' stage, differentiation, and ploidy were good indicators of higher risk of colorectal cancer-related death in patients undergoing curative surgery. Dukes' stage and ploidy were also indicators for recurrence. Thus, routine histopathologic characteristics should be used in combination with quantitative cytologic features for the definition of a relevant prognostic index in colorectal cancer.

Expression of epidermal growth factor receptor and HER-2/neu in normal and neoplastic cervix, vulva, and vagina.

Monoclonal antibodies were used to localize immunohistochemically epidermal growth factor receptor and HER-2/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal growth factor receptor and HER-2/neu both were expressed most strongly by basal keratinocytes. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal growth factor receptor and HER-2/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal growth factor receptor. Staining of 33 of these cancers for HER-2/neu was light, although one patient who presented with distant metastases had heavy staining for HER-2/neu. These data suggest that although overexpression of HER-2/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.

Characterization of four newly established human colorectal adenocarcinoma cell lines from Chinese patients.

Four colon adenocarcinoma cell lines, CC-M2, CC-M3, CC-M4, and CC-M2NM, have been established from surgical specimens of 18 unselected patients without the use of "feeder" cells and additional growth factors (e.g., insulin, hydrocortisone, etc.) in the culture medium. The methods of primary cultivation of tissue explants are described. Studies of determination of morphology, growth curve, plating efficiency, chromosomal analysis, CEA and beta-HCG synthesis, and tumorigenicity, were done to characterize the cell lines. Significant variations have been found in one of the four cell lines, both in vitro and in vivo studies. There are distinct phenotypes in the established cell lines which may be useful in studying the cell differentiation and progression of colorectal cancer.

Changes in glycosaminoglycan synthesis and in heparan sulfate deposition in human colorectal adenocarcinomas.

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

Cellular differentiation and histogenesis of rat glandular stomach cancers.

The gastric and intestinal phenotypic expressions of tumor cells in 18 adenomatous hyperplasias, 33 well-differentiated adenocarcinomas, and 16 undifferentiated adenocarcinomas (4 poorly differentiated adenocarcinomas, 10 signet-ring cell carcinomas and 2 mucinous adenocarcinomas) induced by N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide in the rat glandular stomach were studied by histochemical stainings for mucin and immunohistochemical staining for pepsinogen isozyme 1 (Pg 1). By histochemical staining for mucin [by the paradoxical concanavalin A method, the modified method with labeled peanut lectin, the galactose oxidase-Schiff (GOS) reaction, and the sialidase-GOS reaction] and immunohistochemical staining of Pg 1, gastric cancer cells of each histological group could be clearly classified into a gastric type, including mucous neck cell pyloric gland cell, and surface mucous cell subtypes, and an intestinal type, including goblet-cell, and intestinal absorptive cell subtypes. All tumors examined in this work consisted mainly of gastric-type cells but intestinal-type tumor cells were occasionally found among the gastric-type tumor cells. The incidences of intestinal-type cells in adenomatous hyperplasias (11.1%) and small well-differentiated adenocarcinomas (28.6%) were significantly less (P less than 0.05) than that in large well-differentiated adenocarcinomas (68.4%). The incidence of intestinal-type cells in small undifferentiated adenocarcinomas (25.0%) was also less than that in large ones (58.3%). The present results suggest the occurrence of change of phenotypic expression of tumor cells from the gastric type to the intestinal type during growth of tumors.

Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

Distribution of beta-human chorionic gonadotropin-positive cells in noncancerous gastric mucosa and in malignant gastric tumors.

The authors examined the localization and behavior of beta-human chorionic gonadotropin (HCG)-positive cells in human gastric noncancerous mucosa and in gastric malignant tumors, using immunohistochemistry and the anti-beta-HCG antibody. The beta-HCG-positive cells were located mainly in the antral mucosa and were generally restricted to the neck portion of the pyloric glands, although a few were present in fundic glands of the gastric body. The beta-HCG-immunoreactive cells were found in gastric carcinomas in 53% of the 92 cases examined. These cells were observed more often in advanced carcinomas that were histologically poorly differentiated than in early carcinomas or in well-differentiated tumors, but this prevalence had no statistical significance. The presence of the beta-HCG-positive cells in the gastric carcinomas suggested no appreciable prognostic significance, even quantitatively. In the syncytiotrophoblast-like tumor cells seen in four gastric tumor samples with histologic features of a choriocarcinoma, immunoreactivity to the beta-HCG was striking. There was, however, no recognizable dominance in the number of beta-HCG-reactive cells in the noncancerous mucosa around the tumor.

Lectins and immunohistochemistry of colorectal cancer, its recurrences and metastases.

In 31 patients resected specimens from primary colorectal cancers, corresponding liver metastases and local recurrences were investigated for the staining pattern of lectins (PNL, UEA, WGA, HPA, SBA, RCA) and tissue antigens (CEA, SP, ACT) by immunohistochemistry. Comparison of staining patterns showed a loss of marker expression from normal colonic mucosa to colorectal primary carcinomas, and a tendency to marker loss from the primary tumour to liver metastases. However, even a neo-expression of markers not present in the primary tumour could be observed. For clinical use, serum markers observed in patient follow-up may be valuable even where the findings are negative at the time of primary tumour surgery. In contrast to the heterogenous marker map of primary tumours and metastases, comparison of primary and locally recurrent tumour revealed a staining pattern that was almost always identical. This supports the hypothesis that locoregional recurrences develop from remnant cells of the primary tumour left behind at surgery. There is no support for the thesis that locoregional recurrences arise from mucosal changes at the anastomosis or from suture material.

Metabolism of low-density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells.

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Ovarian adenocarcinomas express fms-complementary transcripts and fms antigen, often with coexpression of CSF-1.

In earlier studies of oncogene expression in ovarian and endometrial neoplasms, the authors reported that high tumor levels of fms-complementary transcripts correlate with high histologic grade and advanced clinical stage presentations. In this communication, they pursue these initial clinicopathologic investigations to demonstrate by in situ hybridization and immunohistochemistry that malignant epithelial cells of 14 of 14 invasive adenocarcinomas of the ovary express fms-complementary transcripts. By Northern blotting and by reverse transcription, followed by polymerase chain reaction amplification, the authors also were able to demonstrate fms transcript expression in several ovarian and endometrial carcinoma-derived cell lines. Because about half (6/14) of the invasive adenocarcinoma specimens were shown to coexpress fms and colony-stimulating factor 1, the authors propose that the expression of this lymphohematopoietic cytokine and its receptor by ovarian adenocarcinomas could contribute to their proliferative and invasive characteristics in vivo.

Expression of a Mr 32,000 laminin-binding protein messenger RNA in human colon carcinoma correlates with disease progression.

Cell surface receptors for laminin may play an important role in tumor migration and metastasis. To evaluate laminin receptor/laminin-binding protein expression in human colon carcinoma, surgical specimens of primary colon cancers and liver metastases were examined by blot hybridization of total RNA with a complementary DNA clone which encodes a Mr 32,000 human laminin-binding protein. The mRNA level of the laminin-binding protein was higher in primary colon carcinoma than in adjacent normal colonic epithelium in 20 of 21 cases. In all 6 cases of colon cancer liver metastases, the laminin-binding protein mRNA level was more than 3-fold greater in tumor than in adjacent normal liver tissue. The tumor/normal ratio of this laminin-binding protein mRNA expression in primary colon cancer has significant correlation with Dukes' classification (P less than 0.001). Our results suggest that mRNA expression of the laminin-binding protein may be a marker of human colorectal cancer progression and biological aggressiveness.

Nuclear DNA distribution pattern of the parenchymal cells in adenocarcinomas of the pancreas and in chronic pancreatitis. A study of archival specimens using both image and flow cytometry.

The nuclear DNA distribution pattern of the neoplastic parenchymal cells of 100 conventionally formalin-fixed and paraffin-embedded specimens from pancreatic adenocarcinomas and from 8 specimens of chronic pancreatitis was assessed by means of image cytometry. All material originated from pancreatic restrictions. Evaluable DNA histograms could be obtained for 77 carcinomas, and clinical data were available for 71 of these. In these 71 specimens, the nuclear DNA ploidy pattern was also investigated by means of flow cytometry. In 76 of the 77 cases, the image-cytometric DNA ploidy pattern obtained showed a "nondiploid" distribution with modal values as high as 8.5 c. In 21 cases, the neoplastic cells showed modal values in the "triploid" region. The analogous 71 flow-cytometric DNA histograms could only be evaluated in 50 cases because of excessively high amounts of background and/or excessively broad peaks. In 47 cases, the nuclear DNA histogram was nondiploid according to both techniques. The patients with carcinomas whose cell nuclei showed a triploid DNA distribution showed a significantly shorter survival time than those with tumor cell populations of nontriploid DNA distribution patterns. In the 8 specimens of chronic pancreatitis, the parenchymal cells were all equipped with nuclei showing diploid DNA distribution patterns.

[Analysis of cellular DNA-RNA content using flow cytometry between primary and metastatic foci in lung carcinoma].

By flow cytometry, cellular DNA-RNA content was analyzed with Acridine Orange staining for primary tumor and the metastatic foci in 12 cases of primary lung carcinoma. Five (42%) of the primary and metastatic foci showed heterogeneous cellular DNA content, with high cellular DNA content of metastatic foci in all cases. Cellular RNA content was significantly higher in metastatic foci than in primary foci, indicating possible higher proliferative activity of the former foci. Three different metastatic foci in one patient showed essentially the same cellular DNA content. From the above it is evident that primary and metastatic foci of lung carcinoma show considerable heterogeneity not only in cellular DNA content but RNA content as well, thus indicating proliferative activity of the metastatic focus. It is consequently advisable to use a combination of various supplementary medications differing in cytocidal effect. Cases with higher cellular DNA content may likely have higher metastatic potential.

Cytokinetic investigation of lung tumors using the anti-bromodeoxyuridine (BUdR) monoclonal antibody method: comparison with DNA flow cytometric data.

In order to investigate the cytokinetics of malignant tumors and non-malignant lesions of the lung, tissue samples from 57 patients affected by non-small-cell carcinoma (NSCLC), small-cell carcinoma (SCLC), and benign and inflammatory lesions have been analyzed using the BUdR monoclonal antibody (MAb) method. This method is based on the preparation, at the time of surgery, of viable monocellular suspensions (using collagenase and DNase treatment) and the concomitant administration of BudR. The percentage of BudR-labelled cells was monitored by fluorescent microscopy using an FITC-labelled second antibody. In NSCLC, each histological group showed a wide range of labelling index (LI) values. On the contrary, SCLC exhibited a more homogeneous kinetic behaviour as evidenced by a narrowly distributed, higher LI. Tumors shown to be diploid by flow cytometry did not show a lower LI than aneuploid tumors. Furthermore, differences were constantly observed between the S-phase percent calculated using BUdR and that calculated using the DNA flow cytometric (FC) histogram, the latter always showing higher S-phase values. In an attempt to study the intra-tumor proliferative heterogeneity, multiple-site sampling was performed. Proliferative heterogeneity seemed to be higher inter-tumor than intra-tumor. Finally, a positive correlation (p less than 0.05) was found between LI and the actual doubling time (DT) of the primary tumor mass, evaluated using sequential radiographs. In conclusion, the present BUdR method can be considered a useful source of relevant information on in vivo cell growth, in parallel to other clinical (DT) and biological (DNA content) approaches.

[Immunocytochemical study of enterochromaffin cells in carcinoma of the large intestine].

In a series of 130 cases of adenocarcinoma of the large intestine, enterochromaffin (EC) cells were detected in 54 cases (41.5%) by immunocytochemistry with anti-chromogranin monoclonal antibody. Among the 54 cases, 30 were found positive for serotonin, 14 for somatostatin, 11 for glucagon, 5 for pancreatic polypeptide, and only one for gastrin. The cases with EC cells (++) or polypeptide positive cells exhibited higher grade of differentiation, earlier stage of tumour extension and higher survival rate than those without EC cells. A significant difference of the EC cell population pattern among different histological grades of the tumours and nonneoplastic mucosa was found. The proportion of hormone, especially polypeptide positive cells was the highest in the mucosa and lowest in the moderately poorly differentiated carcinomas. The incidence, methodology and clinicopathological significance of EC cells found in the tumours are discussed.

[Immunohistochemical study and lectin distribution of thyroid carcinoma originated from follicular epithelium].

97 cases of thyroid carcinoma originated from follicular epithelium were investigated by using histological and immunohistochemical techniques with special reference to lectin distribution. According to the WHO histological typing of thyroid tumours, these cases were divided into three categories as follows: papillary carcinoma of thyroid (PCT) 56, follicular carcinoma of thyroid (FCT) 31 and undifferentiated carcinoma of thyroid (UCT) 10. Results showed that three different kinds of thyroid carcinoma presented various hormone function and distribution of lectins. The positive rate of Tg immunoreactivity was significantly different between these three kinds of tumour, i.e. PCT greater than FCT greater than UCT. Additionally, the positive rate of T4 and T3 immunoreactivity was lower than that of Tg. Some Gastrin, SS and calcitonin positive cells were also recognized in carcinoma of thyroid. Lectin--binding rate of WGA, PNA, SBA and UEA to 97 cases of thyroid carcinoma and 9 cases of normal thyroid tissue revealed that different lectin had a selective binding activity to various types of thyroid carcinoma and normal thyroid cells. From the data obtained, it seemed that the morphological differentiation of thyroid carcinoma was in correspondence with difference of function, and the extent of cell differentiation may be closely related to the biological behavior of the tumour.

[Evaluation of double cancers in relation to previous asbestos exposure].

Ten cases of double cancers (a lung cancer and a stomach cancer) were evaluated in relation to previous asbestos exposure. Ten cases involved male more than 69 years old. Five cases had developed their two cancers simultaneously and other 5 cases had developed their lung cancer after stomach cancer surgery. The lung cancer was their main disease. Four cases had early stage stomach cancers and 5 cases with a stomach cancer had no relapse after surgery. Eight cases had occupational histories of asbestos exposure. Significantly high numbers of asbestos bodies were detected in the autopsied lung in almost all cases. According to the X-ray analysis, almost all asbestos fibers detected in the lung were chrysotile. Additionally, the Brinkman Index (B.I.) of these 10 cases ranged between 700 and 2,000. The combination of asbestos exposure and smoking is thought to be an important factor in the development of such double cancers.

Value of immunohistochemical demonstration of several epithelial markers in hyperplastic and neoplastic endometrium.

The distribution of prekeratin, vimentin, epithelial membrane antigen (EMA), and secretory component (SC) was demonstrated immunohistochemically in 31 patients with adenomatous hyperplasia (AH), 12 patients with atypical adenomatous hyperplasia (AAH), and 39 patients with endometrial carcinoma. Prekeratin was presented in 94% of AHs, 92% of AAHs, and 87% of adenocarcinomas. Vimentin was detected in 68% of AHs, 50% of AAHs, and 37% of adenocarcinomas, showing decreased expression as the lesion progressed to malignancy (P less than 0.05). EMA was detected in 26% of AHs, 67% of AAHs, and 95% of adenocarcinomas (P less than 0.001). SC demonstrated focal and weak expression in 29% of AHs, but showed increased staining intensity in 67% of adenocarcinomas (P less than 0.01). Well-differentiated tumors expressed SC better than poorly differentiated tumors (P less than 0.01). All markers showed a heterogeneous staining pattern and, for a given histologic hyperplastic or neoplastic state, corresponded to several phenotypes. In conclusion, prekeratin seems to be a good marker for epithelial differentiation in hyperplastic endometrium, and EMA is a good marker in neoplastic endometrium. In hyperplastic lesions, the loss of vimentin expression in the absence of secretory changes gives rise to suspicions regarding their benign process. Also, EMA can help in distinguishing between hyperplastic and neoplastic states, while detection of SC may be of help in more precise grading of endometrial carcinoma.