Neurofilament and chromogranin expression in normal and neoplastic neuroendocrine cells of the human gastrointestinal tract and pancreas.

To differentiate neuroendocrine (NE) neoplasms arising at different levels of the gut and pancreas, the authors studied the expression of neurofilament (NF) proteins and chromogranin (CR) in normal and neoplastic NE cells of the human gastrointestinal tract (GIT) (14 ileal/jejunal carcinoids, six appendiceal carcinoids, 11 rectal carcinoids) and pancreas (23 islet cell tumors). Among pancreatic islet cell tumors, those with middle molecular weight (NF-M)-positive cells were more abundant than those with high molecular weight (NF-H)-positive cells; nearly all of these tumors expressed CR. Although NF-M was abundantly expressed in greater than 50% of tumor cells in a subset of these tumors, only one of these tumors exhibited diffuse immunoreactivity with NF-H. Among rectal carcinoid tumors, NF-M and NF-H-positive cells were present in approximately the same number of tumors, yet only diffuse immunoreactivity to NF-H could be detected. Chromogranin immunoreactivity in greater than 50% of tumor cells was present in 74% of islet cell tumors, 93% of ileojejunal carcinoids, and 83% of appendiceal carcinoids, but only in a minority of rectal carcinoids (36%). Although ileojejunal carcinoid tumors rarely expressed NF-M and did not express NF-H, diffuse immunoreactivity with CR was present in nearly all of these tumors. None of the appendiceal carcinoid tumors expressed NF-M or NF-H, yet all of these tumors demonstrated immunoreactivity with CR. Neurofilament immunoreactivity was not detected in normal GIT and pancreatic NE cells, whereas CR immunoreactivity was always present. These results suggest that for NE neoplasms of the GIT and pancreas the differential expression of NF subtypes appears to be related to tumor site; and CR is a marker of most GIT and pancreatic NE neoplasms although NF may discriminate subtypes of GIT and pancreatic NE tumors. Neurofilament subtyping may be useful in the evaluation of the origin of NE tumors presenting as metastatic lesions.

Monoclonal antibody HML-1, a marker for intraepithelial T cells and lymphomas derived thereof, also recognizes hairy cell leukemia and some B-cell lymphomas.

Current views regard monoclonal antibody HML-1 as an exquisite marker for intraepithelial T cells and primary intestinal and cutaneous T-cell lymphomas. We show that HML-1 reacted with 11 of 12 cases of hairy cell leukemia, with 1 of 13 cases of primary gastrointestinal B-cell lymphoma, and with an unclassified large-cell B lymphoma of the thoracic wall. We conclude that HML-1 is not restricted to the T-cell lineage and that the HML-1 antigen is expressed in a small subset of both T- and B-cell neoplasms.

Growth factors and oncogenes in human gastrointestinal carcinomas.

Multi-autocrine loops of the epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), platelet-derived growth factor (PDGF) and TGF beta system are expressed in human gastrointestinal carcinomas. In esophageal and gastric carcinomas, they evidently play an important role in tumor progression. Gastrin, one of the major gut hormones, may also act as an autocrine growth factor for gastric and colonic carcinomas. The HST1 and INT-2 genes, belonging to the fibroblast growth factor gene family, are coamplified in approximately 50% of primary tumors and in all the metastatic tumors of esophageal carcinoma. TGF alpha and EGF are the ligands of the tumor cells that overexpress EGF receptor in esophageal carcinomas. The synchronous expression of EGF and its receptor, as well as TGF alpha and ras p21, is evidently correlated with the depth of tumor invasion, metastasis and prognosis of gastric carcinomas. Amplification of c-erbB-2 and EGF receptor genes has been observed in many metastatic sites of gastric carcinomas regardless of histological type. In addition to TGF alpha and EGF, TGF beta and PDGF A chain produced by tumor cells may stimulate collagen synthesis not only by fibroblasts but also by tumor cells themselves, resulting in extensive progression and diffuse fibrosis of scirrhous gastric carcinomas. Moreover, TGF alpha or EGF and estrogen may also play a cooperative role in the development of scirrhous gastric carcinoma. In colorectal carcinoma, it has been shown that the accumulation of several alterations in ras genes and p53 genes is most important for the conversion of adenoma to carcinoma. Critical genetic changes, including activation of oncogenes, mutation and deletion of tumor suppressor genes and disturbances in transcriptional regulatory sequences, may bring about aberrant expression of growth factors and their receptors in gastrointestinal carcinomas. The understanding of the significance of EGF-related growth factors in tumor progression provides a framework for a biological approach to the therapy of human gastrointestinal carcinomas. 8-Cl-cAMP, which inhibits expression of oncogenes and TGF alpha, may be useful not only for cancer therapy but also for the study of cell differentiation.

[Detection of gut hormone mRNAs by synthetic DNA probes in gut hormone producing tissues].

Synthetic DNA probes were tested as hybridization probes for detecting gut hormone mRNAs. When 4 gut hormones, including gastrin, somatostatin, gastrin-releasing peptide and calcitonin were examined, these probes were shown to be useful for mRNA detection in the tissues producing respective hormones. It was also revealed that there was a good correlation between the concentration of peptides determined by radioimmunoassay and the amounts of mRNAs. This methodology was applied for multiple gut hormone producing tumor with the aim to elucidate the mechanisms responsible for this phenomenon, and demonstrated that the tumor expressed a large amount of mature mRNAs encoding respective hormones. These results indicate that increase of mRNA production is one of the mechanism responsible for multiple gut hormones production by tumor.

Plasminogen activators in endoscopic biopsies as indicators of gastrointestinal cancer: comparison with resection specimens.

In resection tissue samples of colorectal carcinomas, the concentration of urokinase-type plasminogen activator (u-Pa) was found to be significantly higher than in the normal parent mucosal tissue, while there was less tissue-type plasminogen activator (t-PA). u-PA and t-PA were also determined in endoscopic biopsies of colonic and gastric carcinomas, and the results were compared with those of the ultimate resection samples of the same patients, and with the histological evaluation of adjacent biopsies. The ratio of u-PA/t-PA antigen in the biopsies was found to represent a good discriminator between normal and malignant tissue. Nearly all (90%) tumour biopsies had a higher PA antigen ratio than that of the normal tissue biopsies. This discrimination based upon PA antigen measurements in biopsies was similarly efficient in the subsequent resection samples, and showed a good agreement with the histological evaluation. Thus, PA antigen measurements in endoscopic biopsies can be used to detect gastrointestinal malignancy.

Orcein-alcian blue staining: a new technique for demonstrating acid mucins in gastrointestinal epithelium.

Orcein-alcian blue staining, a new method for the simultaneous demonstration of sulphated and sialomucins in gastrointestinal epithelium was compared with the standard high iron diamine-alcian blue technique. Sections were oxidised with potassium permanganate and decolourised in oxalic acid. They were stained with orcein for four hours, differentiated for a few seconds in acid alcohol, and then counterstained with alcian blue for half to one minute. There was a good correlation of results between the two methods. Orcein-alcian blue is a safer, cheaper, and quicker method than high iron diamine-alcian blue which can be safely introduced into routine laboratories for the study of acid mucins in the gastrointestinal diseases.

Gangliocytic paraganglioma.

A study of gangliocytic paragangliomas (GPs) of the gastrointestinal tract from 51 patients showed characteristic microscopic features: epithelioid cells with an endocrine growth pattern, spindle cells, and ganglion cells. Forty-nine tumors were located in the duodenum, 1 in the jejunum, and 1 in the pylorus. Twenty-one patients were female, 28 male, and for two the sex was unknown. The average age at presentation was 54 years (range, 23-83). No patient had a recurrence. No neuroendocrine syndrome was found in any patient or patient's family. Immunohistochemical stains in 33 cases yielded the following (proportion positive): S-100 protein 94%, synaptophysin 94%, neuron-specific enolase 94%, pancreatic polypeptide 88%, somatostatin 75%, chromogranin 72%, neurofilament protein 64%, keratin 52%, leu-enkephalin 48%, serotonin (one case), and gastrin (one case). Antisera usually stained one or two of the three major cell types. Pancreatic-type tissue was identified in or near 28 tumors, including the pyloric and jejunal lesions and two in the distal duodenum. The authors conclude that GP is benign; is not associated with endocrine syndromes; contains autonomic, neural, and endocrine cell types; and is related to pancreatic development.

Plasminogen activators: possible roles in cell proliferation.

Plasminogen activators (PAs) convert plasminogen to plasmin by the cleavage of the Arg-Val bond. There are two distinct types of PA, tissue type (t-PA) released from the endothelial cells of the blood vessels and urinary type (u-PA) released from urinary tubules. u-PA was found to be released from activated macrophages and virally transformed cells. t-PA was also found to be released from breast cancer cells induced by carcinogens or melanoma cells. In structure, t-PA has a finger domain homologous to fibrin-binding domain of fibronectin and a growth factor domain homologous to the epidermal growth factor. u-PA has no finger domain but has a growth factor domain. It is proposed that PA may be important in tumor growth due to the stimulation of tumor cells through binding of growth factor domain to its receptor of tumor cells. Another hypothesis is that PA may activate procollagenase to collagenase, which digests collagen to facilitate tumor growth. We have measured the concentrations of t-PA and u-PA in plasma, urine and tumor tissues of patients with cancer of the digestive tract and patients with uterine or ovarian tumors. The results indicate that the concentrations of u-PA increased in urine, plasma and cancer tissues of patients with cancer of the digestive tracts whereas no increase was observed in t-PA levels. On the other hand, the concentration of t-PA increased mostly in plasma of patients with uterine and ovarian cancers, but t-PA levels in tissues did not increase in patients with uterine and ovarian cancer.

[Determination of cell nuclear DNA content in leiomyoma and leiomyosarcoma of gastro-intestinal tract (G-I)].

Nuclear DNA content was measured by microspectrophotometry in 15 biopsy specimens from patients with G-I smooth muscle tumors (3 leiomyomas and 12 leiomyosarcomas subdivided into 3 groups with 4 cases to each). The mean DNA value increased steadily as follows: leiomyomas (14.39 +/- 0.62 Au); leiomyosarcoma Grade I (19.78 +/- 2.39 Au); Leiomyosarcoma Grade II (26.39 +/- 1.60 Au); leiomyosarcoma Grade III (30.66 +/- 2.39 Au). The difference of DNA value in the 4 groups had statistical significance (P less than 0.05-0.01). These results suggest that microspectrophotometric measurement of nuclear DNA content may serve as an objective quantitative method for diagnosis of G-I tract smooth muscle tumors and classification of leiomyosarcoma.

Diagnostic pathology of gastrointestinal and pancreatic neuroendocrine tumours.

The increased knowledge of the pathobiology of gastrointestinal and pancreatic neuroendocrine tumours and the improved therapeutic possibilities have brought a demand for more precise diagnosis. Although the neuroendocrine tumours can often be tentatively recognized in routinely processed microscopic slides, their more accurate identification requires additional diagnostic procedures. General neuroendocrine markers, such as the argyrophil reaction of Grimelius and immunohistochemistry with application of antibodies against chromogranin A and of neuron-specific enolase are discriminatory staining methods which are used to reveal the neuroendocrine origin of almost all highly differentiated neuroendocrine tumours of the gastrointestinal tract (carcinoids) and pancreas (insulomas). Midgut carcinoids, which predominate among these tumours almost unexceptionally contain serotonin. This biogenic amine can be demonstrated by the argentaffin reaction of Masson, serotonin immunoreactivity or by formalin-induced fluorescence. The characteristic staining pattern of midgut carcinoids is almost invariably preserved in the metastases and can thus be used to reveal a primary midgut carcinoid. The enterochromaffin-like (ECL) cell carcinoids of the body and fundic area of the stomach are argyrophil with Sevier-Munger silver stain. Other neuroendocrine tumours, viz, antral, duodenal and rectal carcinoids and insulomas, should be studied by a battery of relevant peptide hormone antisera for adequate diagnosis. About 50% of all insulin-producing insulomas are endowed with stromal amyloid deposits, which chemically are composed of a peptide designated islet amyloid polypeptide. This molecule has been observed by electron microscopical immunocytochemistry to occur exclusively in the beta-cells and is co-stored with insulin in the beta-cell granules.

Identification of gastroenteropancreatic neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II.

Gastroenteropancreatic human neuroendocrine (NE) cells (normal and neoplastic) were investigated for the expression of the neuroendocrine-specific polypeptides synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II, using immunohistochemistry and immunoblotting. Monoclonal antibody against synaptophysin stained most, and possibly all, of the neuroendocrine cells in both normal and neoplastic tissue. Monoclonal antibody against chromogranin A also stained a high proportion of normal and neoplastic neuroendocrine cells. Immunostaining with polyclonal antisecretogranin I and antisecretogranin II antibodies was detectable in almost all of the normal and neoplastic tissue sections that were analyzed, and it was confined to a smaller population of neuroendocrine cells than that observed for synaptophysin and chromogranin A. Consistent with the immunohistochemical observations, immunoblotting revealed the presence of all four antigens in various tumors. The data show that synaptophysin and chromogranin A, for which monoclonal antibodies are commercially available, may be used as diagnostic markers for human gastroenteropancreatic tumors. Our results also suggest that the development of monoclonal antibodies against human secretogranins I and II will provide additional tools for a refined diagnosis of such tumors.

Cell markers in gastrointestinal stromal tumors.

Stromal tumors of the gastrointestinal (GI) tract have generated considerable controversy about their direction and level of differentiation, particularly about whether the tumor cells are smooth muscle or Schwann cells. In an attempt to characterize these tumors, the immunohistochemical staining patterns of desmin, vimentin, actin, and S-100 protein were studied in 41 GI stromal tumors, using the avidin-biotin method, and compared with normal host smooth muscle and nerve and with esophageal and uterine leiomyomas. Twenty gastric and one rectal tumor stained diffusely with vimentin and actin, but not with desmin, and had scattered strongly S-100-positive cells that might either be trapped Schwann cells or tumor cells. Twenty small bowel tumors stained similarly to the gastric tumors with regard to vimentin, actin, and desmin, but most (17/20) had a unique, strongly positive geographic staining pattern with S-100. No differences in staining were noted between benign and malignant tumors in either gastric or small bowel sites, and most histologic patterns in tumors from similar locations stained similarly. These results suggest that GI stromal tumors are not truly "leiomyomas and leiomyosarcomas," but relatively undifferentiated tumors, with the expression of various antigens depending on their location in the gut.

Selection of a monoclonal antibody reactive with a high-molecular-weight glycoprotein circulating in the body fluid of gastrointestinal cancer patients.

The monoclonal antibody NCC-CO-450 (IgM kappa) was selected by screening of reactivity with high-molecular-weight antigens (Mr greater than 10(6] isolated from ascitic fluid of a colon cancer patient. This antibody detected heterogeneous but predominantly high-molecular-weight antigens in 4 of 6 ascitic fluid samples from gastrointestinal cancer patients by immunoblotting analysis. A sandwich radioimmunoassay was developed in order to examine the serum level of this antigen, and the cutoff value was defined as the mean plus 2 SD of values obtained with sera from normal donors. While 97% (93 of 96) of sera had a negative antigen value in normal donors, 56% (14 of 25) of patients with colorectal carcinoma and 40% (8 of 20) of patients with gastric carcinoma showed a positive antigen value. The distribution of the antigen in sera of patients with various cancers did not show any correlation with the distribution of carcinoembryonic antigen or CA 19-9. From immunohistochemical and biochemical analyses, NCC-CO-450 antigen was characterized as a mucin-like glycoprotein abundant in normal colonic epithelium as well as in carcinomas of the colon, stomach, and pancreas. The immunohistochemical reactivity of NCC-CO-450 was distinct from that of other monoclonal antibodies reported to be useful for serological diagnosis. The epitope recognized by NCC-CO-450 is considered to be an O-linked carbohydrate chain without terminal sialic acid but is different from the known carbohydrate chains, i.e., Lea, Lex, LeY, Tn, sialyl-Lea, and sialyl sugar chain defined by NCC-ST-439 in a competitive binding inhibition assay of monoclonal antibodies. This newly defined antigen is a good example of a normal antigen shed from cancer cells that can be used successfully as a serum tumor marker.

Tumor marker radioimmunoassays in gastric juice. Methodologic drawbacks due to pH variations.

Conflicting data have been reported on tumor marker determination in gastric juice. In the present study the effect of pH variations on both antibody-antigen binding and the immunologic stability of the antigen were evaluated for the radioimmunoassay of carcinoembryonic antigen, CA19-9, tissue polypeptide antigen, and ferritin. A significant inhibition of antibody-antigen binding was constantly found in acidic conditions. Antigen concentration was lower in acidified than in untreated samples, possibly due to the carryover of acidity in the incubation mixture. Neutralization of acidified samples partly improved recovery of carcinoembryonic antigen and CA19-9. Tissue polypeptide antigen and ferritin were not recovered by neutralization in samples with pH less than 4.5, suggesting an irreversible damage of the immunologic characteristics of the two antigens. From the present data we conclude that an accurate validation of methods and a rigorous standardization of sample collection are mandatory for tumor marker determination by radioimmunoassay in gastric juice.

Flow cytometric analysis of paraffin-embedded material in human gastric cancer.

Flow cytometric DNa analysis was performed on formalin-fixed, paraffin-embedded samples obtained by gastroscopic biopsy from 9 patients with histologically normal gastric mucosa (36 specimens) and by radical gastrectomy from 42 cases of human gastric cancer (120 specimens). Ploidy patterns and the distribution of cells in the different cell cycle phases were estimated, and the results were correlated with the histologic and clinical features. All samples of normal mucosa showed a diploid modal DNA content whereas DNA aneuploidy was encountered in 71.4% of the gastric tumors. The correlation between aneuploidy and histologic malignancy grading was statistically significant: aneuploidy was found in 36.4% of highly differentiated (grade 1 and grade 2) tumors and in 75.0% of poorly differentiated (grade 3) tumors (P less than .05). The percentage of cells in S-phase in normal gastric mucosa (median: 5.0%) was lower than that in the tumors (median: 11.3%) (P less than .05). There was a trend for grade 3 tumors to have higher median values (median: 13.4%) than grade 1 and 2 tumors (median: 9.3%); however, this was not statistically significant. An aneuploid DNA pattern was associated with a poorer prognosis, both in early and in advanced stages of gastric tumors, while proliferative activity did not correlate with postoperative survival.

The urokinase type of plasminogen activator in cancer of digestive tracts.

The amounts of urokinase (UK) antigen and tissue plasminogen activator (t-PA) antigen were determined in plasma, urine and tissues of patients with cancer of digestive tracts. Urinary levels of UK, but not those of t-PA increased in patients with cancer, and generally decreased after the removal of cancer by operation. Urinary UK levels kept increasing in patients with recurrence of cancer or with metastasis into liver or peritoneum. Plasma levels of t-PA, but not those of UK decreased in patients with cancer. When the amounts of UK were compared in cancer tissues and their adjacent normal mucosal layer, cancer tissues always had higher levels of UK, but t-PA levels were same between tumor tissues and normal mucosa. The results suggest that the type of plasminogen activator was UK-type in cancer of digestive tracts.

Expression of the human transferrin receptor in subrenal capsule assay in the mouse.

The expression of a cell proliferation marker, the human transferrin receptor, was studied in ten human gastrointestinal tumors prior to and after implantation under the renal capsule in the mouse (1-6 days). These data were compared to the increase in tumor size in situ, and to the infiltration of inflammatory cells. All tumors studied expressed the transferrin receptor prior to implantation. Forty of 47 implants expressed the receptor, the strongest expression occurring on day 4, accompanied by a reorganization of tumor tissue to a morphology similar to that before implantation. On days 5 and 6 the expression of the transferrin receptor declined. Implants showed maximal increase in size on days 1 and 2, decreased in size on days 3 and 4, and increased again on days 5 and 6. The increase in the size of the implants on days 5 and 6 was accompanied by considerable infiltration of inflammatory cells, and was probably mostly a result of invading host cells and inflammation. If size alone is used as a criterion for tumor proliferation in this subrenal capsule assay, day 4 seems to be the most appropriate for evaluation. This is supported by a strong expression of a proliferation marker, the human transferrin receptor, during this time.

[A monoclonal antibody, KM10 reactive with human gastrointestinal cancer].

A monoclonal antibody, KM10 (IgG1) was produced by fusing spleen cells from a human gastric cancer cell-primed BALB/c mouse with the murine myeloma cell line X63-Ag8-653. The antibody reacted strongly with the plasma membrane of human gastro-intestinal carcinoma. Sections of both malignant and benign tissues were tested with immunoperoxidase. Ten of 10 (100%) large intestine cancers, 26 of 31 (84%) gastric cancers, 5 of 7 (71%) pancreatic cancers and 3 of 3 (100%) ampulla of Vater cancers reacted positively. Moderate or little reactivity was observed with normal human tissues and carcinomas of the liver, mammary, thyroid and adrenal glands. According to a study of the distribution of 125I-labeled KM10 in nude mice bearing human gastric cancer, KM10 selectively localized in tumor tissue rather than normal tissue. Whole-body autoradiography also supported such selective distribution. By enzyme treatment and Western blot analysis, the antigenic determinant of KM10 antigen was demonstrated to be protein with a MW of about 240,000.

Neuroendocrine tumors of the gastrointestinal tract.

Neoplastic proliferations of neuroendocrine cells (NE) may occur throughout the entire GI tract but affect particularly appendix and ileum ("midgut carcinoids"), rectum ("hindgut carcinoids"), as well as stomach and the duodenum ("foregut carcinoids"). Only more exceptionally, they arise in the esophagus, jejunum and colon. The NE tumors encompass a heterogeneous gross and microscopic structural spectrum, ranging from inconspicuous microproliferations ("mucous membrane nevi") to bulky tumor masses. Their growth patterns are usually characteristic and easily recognized. In doubtful cases their NE differentiation becomes established by a characteristic silver affinity, by the ultrastructurally observed presence of characteristic "endocrine" secretion granules, and by immunohistochemically detectable occurrence of "pan-NE markers" (neuron-specific enolase, chromogranins, and synaptophysin), biogenic amines (mainly serotonin), and neurohormonal peptides. Foregut carcinoids usually contain serotonin, gastrin, and somatostatin, midgut carcinoids often only serotonin and tachykinins, whereas the hindgut carcinoids as a rule are multihormonal with a wide spectrum of hormonal peptides, including even insulin. Most GI NE tumors are found in the appendix (50%) and the ileum (30%). Practically all (98%) of the appendiceal NE tumors are benign. They have recently been proposed as arising from apparently Schwann-cell-related NE cells in the submucosa, whereas the ileal--and probably also all the other non-appendiceal NE tumors--are derived from the totipotential cells in epithelial crypts of the mucosa. Among the ileal NE neoplasms a large number can metastasize and result in a fatal outcome. The ability to metastasize is related to the size and to the multiplicity of the primary tumors at the time of initial diagnosis and, to some extent, to their histopathologic growth pattern. Now, some relationship between the prognosis and the cytochemically assessed nuclear DNA content of the NE tumor cells has also been established; not less than about 1/4 to 1/3 seem to be aneuploid. Almost 90% of the rectal carcinoids are benign. Exceptionally, a highly malignant NE neoplasms can arise from the colon/rectum--as well as from the esophagus--composed of NE cells of small and intermediate size. The NE tumors of the stomach are often composed of ECL (enterochromaffin-cell-like) cells; such ECL cell carcinoids are related to atrophic gastritis with pernicious anemia; experimentally, they can be induced by hypergastrinemia in rats. Duodenal carcinoids often contain psammoma bodies and can be associated with neurofibromatosis.