Cytometric and histologic predictors of prognosis in ampullary carcinoma treated with pancreatico-duodenectomy

The association between several clinical, histologic and karyometric variables and the survival rates of patients with ampullary carcinoma was examined. Cases were limited to those treated exclusively by pancreaticoduodenectomy for which follow-up data was available, eliminating those cases with deaths due to other causes. The histologic type was classified as papillary, intestinal or mixed while the differentiation was recorded as well, moderate or poor. The stroma was categorized as scanty, moderate or abundant, and the tumor stage was evaluated according to Martin's classification. High-resolution morphometric and microphotometric (DNA content) evaluation of Feulgen-stained nuclei was performed using the microTICAS system. Statistical analyses were performed to examine the relationship between these variables and survival. Neither the tumor stage nor the presence of positive lymph nodes was a significant prognostic indicator, nor was the degree of differentiation or the amount of stroma. However, the survival showed a significant association with several karyometric variables and with the histologic type. Specifically, aneuploid DNA ploidy profiles, higher mean ploidy values and larger nuclei were associated with a lower survival rate. Short-term survivors (less than five years) had a mean ploidy of 2.8N, a mean 5N exceeding rate of 8.3% and a mean nuclear area of 41 sq microns, while long-term survivors (greater than or equal to five years) had corresponding means of 1.9N, 0.6% and 26 sq microns. These differences are all significant at a two-tailed significance level of less than .05 using a separate variance estimate t-test. In addition, papillary tumors showed a better prognosis than did intestinal or mixed tumors (Breslow P less than .04 and Mantel-Cox P less than .009).     

Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells

Abstract. By fusion of nullipotent embryonal carcinoma F9 cells with certain somatic cells freshly collected from differentiated tissues such as thymus and lens, pluripotent hybrid cell lines were obtained. They exhibited a wide spectrum of differentiation, including neural tubes, cartilages, skeletal muscles, ciliated epithelia and others, in solid tumors formed after injection into syngeneic mice. Cells from these tumors differentiated into several cell types when cultured in vitro. A possibility of the introduction of genes to code the factors for regulating differentiation into F9 cells by fusion is suggested.     

Epidemiological Study of Mammary Tumors in Female Dogs Diagnosed during the Period 2002-2012: A Growing Animal Health Problem

Epidemiological studies enable us to analyze disease behavior, define risk factors and establish fundamental prognostic criteria, with the purpose of studying different types of diseases. The aim of this study was to determine the epidemiological characteristics of canine mammary tumors diagnosed during the period 2002-2012. The study was based on a retrospective study consisting of 1,917 biopsies of intact dogs that presented mammary gland lesions. Biopsies were sent to the Department of Pathology FMVZ-UNAM diagnostic service. The annual incidence of mammary tumors was 16.8%: 47.7% (benign) and 47.5% (malignant). The highest number of cases was epithelial, followed by mixed tumors. The most commonly diagnosed tumors were tubular adenoma, papillary adenoma, tubular carcinoma, papillary carcinoma, solid carcinoma, complex carcinoma and carcinosarcoma. Pure breeds accounted for 80% of submissions, and the Poodle, Cocker Spaniel and German Shepherd were consistently affected. Adult female dogs (9 to 12 years old) were most frequently involved, followed by 5- to 8-year-old females. Some association between breeds with histological types of malignant tumors was observed, but no association was found between breeds and BN. Mammary tumors in intact dogs had a high incidence. Benign and malignant tumors had similar frequencies, with an increase in malignant tumors in the past four years of the study. Epithelial tumors were more common, and the most affected were old adult females, purebreds and small-sized dogs. Mammary tumors in dogs are an important animal health problem that needs to be solved by improving veterinary oncology services in Mexico.     

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.     

IN VIVO STUDY ON THE PROCESS OF SOLID TUMOR FORMATION FROM EMBRYOID BODIES OF MOUSE TERATOCARCINOMA

Embryoid bodies of mouse teratocarcinoma OTT 6050 were studied with special reference to their growth and differentiation in vivo . They were grown either in the peritoneal cavity or in the lung. When injected intraperitoneally, embryoid bodies doubled their number every three days. Some of them attached to a small intra-peritoneal fat body and were soon surrounded by mesenchymal cells of host origin. They grew, fused with each other and became large solid tumors which contained many differentiated tissues. When injected intravenously, almost all the embryoid bodies lodged in the lungs and individually grew into discrete solid tumors which doubled in volume every 2.9 days. After about thirty days, some tumors were composed of only one type of tissue while others contained several types of tissues. The possible mechanisms for the uni- and multi-tissue types of differentiation are discussed.     

Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells.

Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.     

C-erbB-2 oncoprotein in gastric carcinoma: correlation with clinical stage and prognosis

The aim of this study was to investigate the expression of the oncogene c-erbB-2 in gastric tumors. Immunohistochemical study of the expression of c-erbB-2 was performed in formalin-fixed, paraffin-embedded sections from 82 gastric adenocarcinomas using polyclonal antibody. c-erbB-2-positive immunostaining was observed in 37 (45%) tumors. Positive staining was detected in 63% of well differentiated, 46% of moderately differentiated and 80% of papillary adenocarcinomas. In poorly differentiated adenocarcinomas, positivity for c-erbB-2 was observed in 21 %. According to the Lauren classification, a higher frequency of c-erbB-2 positive staining was observed in intestinal type tumors (70%). During the follow-up period 43% of the patients with c-erbB-2 oncoprotein-negative tumors and 45% of the patients with c-erbB-2 oncoprotein-positive tumors died. There was no significant association between c-erbB-2 staining and sex, age, clinical stage, tumor grade, histological type or survival rates. In conclusion, almost half of the gastric cancers were positive for c-erbB-2. Nonetheless, the expression of c-erbB-2 oncoprotein did not play a role in prognosis.     

Spontaneous hepatocellular neoplasms and hepatic hemangiosarcomas in several strains of mice

The incidence of spontaneous liver tumors, ratio of hepatocellular adenomas to carcinomas, degree of differentiation of hepatocellular carcinomas, and incidence of pulmonary metastases in several strains of mice from different experiments were examined. Hepatocellular tumors were rare in all strains before 12 months of age and then increased with age. They were more common in male than in female mice. Pulmonary metastases were rare before 24 months of age and occurred in 10 of 221 mice with hepatocellular carcinomas (4.5%); their incidence in different strains was not strikingly different. Hepatic vascular tumors were less common than hepatocellular tumors. No tumors of bile duct origin were observed in these mice.     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.     

Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells.

Three of 16 human gastric adenocarcinoma samples, maintained as solid tumors in nude mice, were found to carry amplified c-myc genes. In two samples with a high degree of c-myc DNA amplification (15- to 30-fold), double minute chromosomes were observed in karyotype analysis. The level of c-myc RNA was markedly elevated in a rapidly growing and poorly differentiated tumor, whereas it was only slightly elevated in a slowly growing and more differentiated tumor.     

Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer.

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.     

Comparative study of tumor angiogenesis and immunohistochemistry for p53, c-ErbB2, c-myc and EGFr as prognostic factors in gastric cancer

Gastric cancer (GC) continues to be a highly aggressive malignancy with poor prognosis and low survival rates. The survival of patients with GC depends mainly on the stage of the disease, with early GC having a 5 year survival of 90-100% and advanced tumors a 5 year survival of 15-25%. The role of other prognostic factors in these tumors is still under investigation. 28 gastric dysplasia, 45 Early GC and 98 Advanced Gastric Cancers were evaluated for expression of the oncogenes p53, c-ErbB2, c-myc and the EGFr in paraffin-embedded material utilizing Avidin-Biotin immunohistochemistry techniques. In 34 cases of GC microvessel density (MVD) was determined in CD34 stained sections. Statistical correlations with stage, histologic type, differentiation degree, location, size, ploidy patterns and overall survival were done. The Mantel-Cox test was performed to evaluate which factors had an independent prognostic value. Both, tumor angiogenesis and p53 protein expression were statistically associated (95% confidence intervals) with overall survival in patients with GC. p53 protein expression was also correlated with cardial location, nodal involvement and tumor stage. c-ErbB2 may recognize a group of highly aggressive well differentiated adenocarcinomas with worse prognosis. c-myc was also significantly enhanced in well differentiated tumors. EGFr showed no significant associations. Mantel-Cox was performed to compare the prognostic value of tumor stage, p53 protein expression and tumor angiogenesis. Tumor angiogenesis was the most important prognostic indicator to predict overall survival in our series. p53 expression was not independent and did not provide additional prognostic information to tumor stage. Our study suggests that angiogenesis as demonstrated by microvessel counts in CD34 stained sections is a significantly important prognostic factor for predicting survival in gastric cancer.     

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.     

Characterization of two TCR transgenic mouse lines specific for herpes simplex virus

To better understand the T cell-mediated processes involved in the immune response to herpes simplex virus type 1 (HSV-1)infection, two HSV-specific T cell receptor (TCR) transgenic mouse lines were produced. These mice (gBT-I.1 and gBT-I.3) are MHC class I-restricted and specific for the immunodominant peptide from HSV glycoprotein B (gB), gB498-505. Although derived from the same clone, the mice differ in the chromosomal location of the TCR transgenes and show marked differences in TCR alpha/beta expression on both CD4+ and CD8+ cells in the thymus. Despite this, peripheral CD8+ Tcells from both mice express equally high levels of the transgenic TCR and bind the KbgB498-505 tetramer to the same degree. In concordance with this, both were shown to respond equally well in vitro upon stimulation with the gB498-505 peptide or HSV-infected cells. These data show that selection of broadly equivalent peripheral T-cell subsets can occur in the presence of distinctly different thymic T-cell subsets.     

Ciliated cells in papillary adenocarcinomas of Barrett's esophagus.

Eighteen surgical specimens having an adenocarcinoma arising in Barrett's esophagus were reviewed, and special attention was paid to the presence of ciliated cells. The tumors were classified as glandular (9), papillary (4), diffuse (3) and mixed (2) types. Ciliated cells were observed in one specimen, in cystically dilated glands in Barrett's mucosa adjacent to a papillary adenocarcinoma. Ciliated tumor cells were found in three of the four papillary adenocarcinomas. The fourth papillary tumor, 1 mixed papillary-diffuse adenocarcinoma and the remaining 13 adenocarcinomas had no ciliated cells. Thus, the presence of cilia in exfoliated tumor cells from the esophagus should raise the suspicion of a papillary adenocarcinoma arising in Barrett's mucosa.     

Precancerous stem cells have the potential for both benign and malignant differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: II. A conversion coupling of tumorigenicity and a glial property

RT4 is a neurotumor induced by ethylnitrosourea injection of a newborn BDIX rat. We demonstrated previously that heterogeneity in early cultures of RT4 tumor cells can be regularly reproduced in cultures of clonal stem cells (cell type conversion). Our previous studies included morphology, differentiation of neural properties, and chromosome number of “tumor-derived” and “stem cell-derived” differentiated cells. In this paper, these two sets of differentiated cells were examined further for three additional parameters, all of which are related to malignancy. The stem cell (AC) and one type of differentiated cell (D) cause tumors when subcutaneously injected into syngeneic animals, while the other two types (B and E) do not. The amounts of a 250,000 molecular weight cell surface protein, which is probably equivalent to LETS protein (large external transformation-sensitive protein) of hamster and mouse, and the levels of plasminogen activator were examined as possible markers of malignancy. As anticipated, nontumorigenic cells generally have a large amount of the 250,000 molecular weight cell surface protein and are low in plasminogen activator activities, whereas the reverse is true for tumorigenic cells. This supports the idea that B and E cells are nontumorigenic revertants. The cell type conversion phenomenon of RT4 neurotumor and the differentiation of mouse teratoma and myeloid leukemic cells share a number of similarities, but differ in that differentiated RT4 cells can propagate in vitro even after loss of tumorigenicity. The concomitant expression of tumorigenicity and the S100 protein production of the D cell, or of nontumorigenicity and B and E cell differentiation upon the conversion of the stem cell, may suggest a regulational coupling between the tumorigenicity and the expression of a glial protein (S100 protein) in D cells.