Heterogeneity of DNA distribution in diploid cells: a new predictive discriminant factor for solid tumour behaviour.

Spatial nuclear DNA heterogeneity distribution of Feulgen-stained DNA diploid cells was studied by image cytometry in voided urine of 119 patients without bladder tumour (n = 20) and with initial (n = 23) or previous (n = 76) diagnosed bladder tumour. For each patient, repetitive DNA measurements were performed during 1-4 years of follow up. Only cells of diploid DNA histograms and diploid subpopulations of aneuploid DNA histograms were used for analysis. DNA heterogeneity distribution of these diploid cells was quantified by statistical parameters of each nuclear optical density distribution. Discriminant analysis was performed on three groups of DNA histograms. Group A (n = 44): aneuploid DNA histograms of patients with bladder tumour. Group D (n = 55): 38 diploid DNA histograms of the 20 patients without bladder tumour (subgroup D1) and 17 diploid DNA histograms of patients with a non-recurrent bladder tumour (subgroup D2). Group R (n = 27): diploid DNA histograms of patients with bladder tumour recurrence. No statistically significant discriminant function was found to separate D1 and D2. However, the first canonical discriminant function C1 differentiated diploid cells of diploid DNA histograms (group D and group R) from diploid cell subpopulations of aneuploid DNA histograms (group A). Mean C1 values were 1.06, 0.84 and -1.45 for groups R, D and A, respectively. The second canonical discriminant function C2 differentiated diploid DNA histograms of patients with bladder tumour recurrence (group R) from diploid DNA histograms of patients without bladder tumour or without bladder tumour recurrence (group D). Mean C2 values were 1.78 and -0.76 for groups R and D, respectively. In 95% confidence limit, the rate of rediscrimination using the two first canonical discriminant functions C1 and C2 were 86.4, 74.5 and 74.1% for groups A, D and R, respectively. Percent of "grouped" cases correctly classified was 78.6%. Thus spatial DNA heterogeneity distribution of diploid cells seems to quantitate probable genetic instability as a function of clinical evolution such as tumour recurrence, and suggests the possible presence of aneuploid stemlines in a heterogeneous tumour, even if a diploid DNA histogram is observed in a single sample. From standardized C1 and C2 canonical discriminant function coefficients, a DNA heterogeneity index (2c-HI) is proposed to characterize diploid cells providing a descriptive and predictive discriminant factor for solid tumour behaviour.

Immunohistological characterization of mucin epitopes by pre-treatment of gastro-intestinal sections with periodic acid.

Periodate pretreatment of paraffin sections of ethanol-fixed gastrointestinal mucosae was used to characterize the carbohydrate or peptidic nature of mucin epitopes by immunoperoxidase. Immunoreactivity of monoclonal antibodies (MAbs) against histo-blood group related carbohydrate epitopes such as A, Lea, Lec, Sialosyl Lea, H type 2, I, T, Tn and sialosyl Tn dramatically decreased or even disappeared after periodate pretreatment of deparaffinized sections. In contrast, the immunoreactivity of MAbs against peptide mucin epitopes such as the gastric M1 mucin epitopes was almost unaffected by this treatment. Moreover, periodate treatment revealed cryptic peptide M1 mucin epitopes and the peptide MUSE11 epitope associated with the 20 amino acid tandem repeat (PDTRPAPGSTAPPAHGVTSA). An increase of cross-reactions of anti-human M1 MAbs with gastric epithelium of different vertebrate species was detected with periodate treatment. Our results suggest that this method can be useful for preliminary characterization of the biochemical nature of mucin epitopes (peptidic or saccharidic) and for demasking peptidic tumour markers which are hidden by saccharide molecules in normal tissues.

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.

Distribution of oncofetal fibronectin in human mammary tumors: immunofluorescence study on histological sections.

Two types of human fibronectin have been detected by the reactivity of specific monoclonal antibody FDC-6: (a) those present in normal plasma and adult tissues, which do not react with FDC-6 (normal fibronectin or norFN); and (b) those present in fetal tissues and in tumoral cell lines, which do react with FDC-6 (oncofetal fibronectin or onfFN) (H. Matsuura and S. I. Hakomori, Proc. Natl. Acad. Sci. USA, 82: 6517-6521, 1985). We compared the distribution of norFN and onfFN in normal breast tissue (15 samples), breast fibroadenoma (ten samples), and breast adenocarcinoma (80 samples), using an immunofluorescence technique with monoclonal antibody FDC-6. A polyclonal antiserum against human normal fibronectin was used as a control. While norFN was diffusely present in normal gland and in benign and malignant tumors, onfFN was absent in normal gland and in benign tumors. In carcinomas, however, its presence was frequent, as we found it in 60% of cases. Among classical prognosis factors in breast carcinomas, onfFN distribution was significantly correlated with histological grade. Indeed, the presence of FDC-6 labeling was significantly linked with intermediary and high malignancy grades, while its absence was significantly linked with low malignancy grade. onfFN could be considered a marker of the neoplastic state; its immunohistological detection may represent a new prognosis factor in breast carcinomas.

Comparative distribution of fibronectin and vitronectin in human breast and colon carcinomas. An immunofluorescence study.

Distribution of two extracellular matrix components, vitronectin and fibronectin, was studied by immunofluorescence in 40 breast infiltrating duct carcinomas and in 15 colonic adenocarcinomas. Blood vessels, especially in peritumoral colonic submucosa, gave markedly different images after staining by antivitronectin antibody and antifibronectin serum. The former exclusively decorated the elastic laminae, whereas the latter gave a reaction much closer to the endothelium, probably localized at the basement membrane of the endothelium. Strong labeling of colonic and mammary stroma carcinomas by polyclonal antiserum against fibronectin was observed, whereas monoclonal antibody against vitronectin gave much more restricted labeling in the stroma of these tumors. In the stroma of colonic carcinomas, vitronectin was often situated in areas in which elastic fibers were visualized by orcein counterstaining. In breast stroma carcinomas it was always localized only in elastosis areas. Thus, it is likely that antivitronectin binds with glycoprotein(s) associated with elastic fibers. However, vitronectin staining also was seen in areas in which no elastic fibers were characterized by the orcein method.

Ultrastructural changes in acinar cells of hamster pancreas in chemically induced carcinogenesis.

Ultrastructural changes arising in the pancreas of the Syrian golden hamster after treatment with N-nitrosobis (2-oxopropyl) amine (BOP) were studied at short intervals. Alterations were found in acinar cells i.e. loss of zymogen granules, dilatation of granular endoplasmic reticulum, depolarization, irregular nucleus and separation of lateral surfaces (intermembranary spaces). As a result, the compact morphology of normal acini switched towards a new structure resembling a pseudo-ductule. Such alterations occurred from the 3rd month and preceded tumor formation. It is noteworthy that ducts and islets of Langerhans appeared unaltered in all instances. These results are consistent with the hypothesis that BOP induced pancreatic adenocarcinoma in hamsters originates in the acinar cell.

Protein synthesis inhibition and cardiac lesions associated with deoxynivalenol ingestion in mice.

Deoxynivalenol (DON), an occasional contaminant of foodstuffs, has been implicated in outbreaks of mycotoxicosis. Balb-c mice that had ingested 0.35 mg/kg of DON showed a drastic decrease in food intake and concomitant loss of weight. Severe depletion of the lymphoid organs and liver were also observed. Cardiac lesions, appeared as calcified pericarditis foci in young animals fed a diet contaminated by 10 to 20 ppm of DON for a period of a few weeks. DON inhibited protein synthesis. This inhibition occurred at lower doses for the heart than for the other organs. This preferential effect on cardiac tissue correlated with the cardiotoxicity observed.

Expression of oncofoetal pancreatic antigens in hamster adult pancreas during experimental carcinogenesis.

Foetal acinar components associated with the development of the hamster pancreas have been previously defined with the aid of an antifoetal pancreas serum. In immunohistology this antiserum also stained malignant ductal cells in N-nitrobis (2-oxopropyl) amine (BOP)-induced pancreatic adenocarcinoma. It did not stain adult pancreas structures including acini, ducts and islets of Langerhans. In this study, re-expression of foetal acinar antigens was disclosed before formation of tumours. Adenocarcinomas were not detected by conventional histology before the 24th week following initiation of the chemical treatment. However, staining with the antiserum was observed from the 7th week appearing in the apex of some acini cells having an almost normal histological appearance. Later, foetal acinar expression was frequently associated with evident morphological alterations in acini like dyskaryosis, enlarged cytoplasm or lumina. Staining of ducts with marked atypical epithelium and (as already reported) of neoplastic ducts was also observed. It was not detected in other pancreatic lesions viz. cystadenomas, mucoid glands and regular hyperplastic ducts. Acinar dedifferentiation as assessed by expression of foetal components preceded formation of tumours in all instances.

Fetal gastric and small intestine pattern of intestinal mucus antigens in human gastric carcinomas.

The present immunohistological study was performed to investigate the expression of intestinal mucus-associated antigens in the different histological types of gastric carcinoma according to the classification of Laurèn and the WHO classification. We used the following antigenic markers: M3, present in all the goblet cells of the whole small and large intestine; M3SI, expressed in all the goblet cells of the small intestine, but only in some of them of the large intestine; M3D, mainly produced in the upper small intestine; and M3C, specific for colonic mucus cells. All these antigens were found to be similarly expressed in both Laurèn's intestinal and diffuse types. Nevertheless, M3 and M3C appeared to be more largely produced in carcinomas showing well-differentiated cells (tubulopapillary, mucinous, and signet ring cells according to the WHO classification). Our results evidenced the occurrence of two main fetal antigenic patterns in gastric carcinomas, one of the gastric type (M3SI produced to a larger extent than M3 and M3C) and the other one of the small intestinal type (M3 and M3SI more largely expressed than M3C). On the basis of their similar antigenic pattern, the histogenesis of carcinomas showing the fetal small intestinal antigenic profile may be associated with intestinal metaplasia. On the other hand, carcinomas with the fetal gastric pattern may originate from undifferentiated stem cells of the gastric mucosa. Thus, such immunohistological studies could lead to a better understanding of the histogenesis of gastric carcinomas.