Correlation between spontaneous and experimentally induced tumors in female BALB/c mice in a large 2-acetylaminofluorene study

This investigation studied the incidence of both spontaneous and induced neoplastic lesions in 6,938 BALB/c female mice receiving 0, 75, 100 or 150 ppm of 2-acetylaminofluorene (2-AAF). The mice were maintained under barrier-type, specific pathogen free/defined flora (SPF/DF) conditions, and were serially sacrificed at 9, 12, 14, 15, 16, 17, 18, 24, and 33 mon. The most frequently observed neoplasms, the incidence of which averaged over 20% and did not increase with the administration of 2-AAF, included lymphomas, alveolar-bronchiolar tumors and uterine polyps. Other common but less frequently observed neoplasms, which were also not increased by the administration of 2-AAF, included adrenocortical adenomas, angiosarcomas and ovarian, mammary and Harderian gland tumors. These spontaneous tumors accounted for almost 95% of the tumors in the control group. Conversely, induced hepatocellular and urinary bladder tumors increased in incidence with the dose level and length of administration of 2-AAF. The number of tumors per animal increased with both age and dose level of 2-AAF. After 400-500 days of life the increased incidence of induced urinary bladder and hepatocellular tumors resulted in a statistically significant increase in the ratio of tumors per animal between controls and the 150 ppm group and after 18 mon at 100 ppm. The incidence and ratio of tumors in the treated groups exclusive of bladder and liver tumors were not statistically different with those of the control group. The administration of 2-AAF did not appear to induce tumors in female BALB/c mice, except for bladder and liver tumors, and neither promoted nor inhibited the development of spontaneous tumors.     

Hepatoblastomas in mice in the US National Toxicology Program (NTP) studies

Over the last 8 years, a 5-fold increase in the incidence of mice with spontaneous hepatoblastomas and a moderate increase in the incidence of chemically induced hepatoblastomas in B6C3F1 mice occurred in 2-year NTP studies compared to the previous 7 years. There was a positive association between an increased incidence of mice with hepatoblastoma and an increased incidence of mice with hepatocellular tumors in the treated mice. The rate of pulmonary metastases for hepatoblastoma was similar to that of pulmonary metastasis for hepatocellular carcinomas. Although a variety of chemicals caused an increased incidence of mice with hepatoblastoma, there was no apparent association between a specific chemical structure or a biological class of compounds and their capacity to induce hepatoblastomas. Hepatoblastomas frequently arose within hepatocellular carcinomas or adenomas and were induced by the same compounds that induced hepatocellular neoplasms. Therefore, it seems reasonable to combine the incidence of mice with hepatoblastomas and the incidence of mice with hepatocellular carcinomas in hazard identification studies.     

Morphologic characteristics of benign and malignant renal cell tumors in control and 2-acetylaminofluorene-treated BALB/c female mice.

A total of 108 mice with renal cell tumors were obtained from a total of 25,916 control and 2-AAF-treated BALB/c female mice. A total of 119 renal cell adenomas and 37 renal cell carcinomas from the 108 mice were classified morphologically. The 119 renal cell adenomas were divided into cystic (32%), cystopapillary (29%), papillary (24%), and solid (15%). The 37 renal cell carcinomas were divided into tubolosolid (16%), tubolopapillary (49%) and tubulopleomorphic (35%). Pulmonary metastases occurred only in the tubulopleomorphic adenocarcinomas. In mice having renal cell tumors and surviving less than 650 days, the renal cell carcinomas were usually multiple and renal cell adenomas often coexisted in the same kidney. In mice having renal cell tumors and surviving more than 650 days, the renal cell carcinomas usually consisted of single large tumors without coexisting renal cell adenomas. The results suggest that the renal cell adenoma may progress to renal cell carcinoma. The various dose levels of 2-AAF did not significantly affect the incidence of renal cell tumors.     

Comparative effects of carcinogens on the induction of placental glutathione S-transferase-positive liver nodules in a short-term assay and of hepatocellular carcinomas in a long-term assay

The dose-dependent effects of three hepatocarcinogens were investigated by measuring the number and area of glutathione S-transferase placental form (GST-P)-positive foci and nodules appearing in the liver under short-term conditions (Experiment I) and evaluating the incidence of hepatocellular carcinoma after long-term chronic administration (Experiment II). For these purposes, three different doses of 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethy-laminoazobenzene (3'-Me-DAB), and DL-ethionine (ethionine) were given to male F344 rats for 6 weeks after a single injection of diethylnitrosamine (DENA) in Experiment I or for 104 weeks without initiation by DENA in Experiment II. In Experiment I, the induction of GST-P-positive foci and nodules by 2-AAF and 3'-Me-DAB was clearly dose-dependent. In contrast, ethionine showed enhancing effects inducing GST-P-positive foci and nodules only in groups given the highest dose level. Similarly, in Experiment II, induction of hepatocellular carcinomas by 2-AAF and 3'-Me-DAB was clearly dose-dependent, whereas liver neoplasms were only induced by the highest dose level of ethionine. These results indicate that degree of induction of GST-P positive foci and nodules in a short-term in vivo test for liver carcinogens corresponds with the incidences of hepatocellular carcinomas revealed in a long-term in vivo assay.     

Comparative prevalence,multiplicity, and progression of spontaneous and vinyl carbamate-induced liver lesions in five strains of male mice

The overall and age-specific prevalences and multiplicities of spontaneous and chemically induced hepatocellular neoplasia were compared among male B6D2F1, B6C3F1, C3H (C3H/HeNCr1 MTV-), B6CF1, and C57BL/6 (C57BL/6NCr1) mice following a single intraperitoneal injection of 0.03 microM vinyl carbamate (VC)/g body weight or vehicle alone at 15 days of age. Additional groups of B6C3F1, C3H, and C57BL/6 males received 0.15 microM VC/g body weight at 15 days of age. For male B6D2F1, B6C3F1, C3H, B6CF1, and C57BL/6 mice, the estimated overall prevalences (and multiplicities) of hepatocellular adenomas or carcinomas in vehicle controls were 14.1% (0.19), 12.3% (0.15), 8.2% (0.10), 7.2% (0.09), and 2.4% (0.02), respectively. The analogous estimates in the low-dose group were 59.2% (1.19), 72.9% (4.07), 48.6% (1.99), 22.8% (0.29), and 43.9% (0.82). Analogous estimates for B6C3F1, C3H, and C57BL/6 mice in the high-dose group were 45.3% (4.29), 59.7% (6.63), and 46.8% (1.74), respectively. Age-specific multiplicity estimates suggested a progression from altered hepatocellular foci (AHF) to hepatocellular neoplasms. Further evidence of progression was provided by the temporal occurrence of hepatocellular adenomas before carcinomas, and the apparent origination of carcinomas within adenomas. Pulmonary metastases were observed in many of the mice with hepatocellular carcinomas. These findings confirm previous observations of strain differences in liver neoplasm response, suggest a progressive development from AHF to adenomas, and ultimately to carcinomas, and show sensitivity to VC-induced hepatocarcinogenesis in all 5 strains.     

Bromoethane, chloroethane and ethylene oxide induced uterine neoplasms in B6C3F1 mice from 2-year NTP inhalation bioassays: pathology and incidence data revisited

Chloroethane, bromoethane and ethylene oxide represent a unique set of chemicals that induce endometrial neoplasms in the uterus of B6C3F1 mice following an inhalation route of exposure. The results of the NTP's chronic bioassays with these three compounds resulted in an unusually high incidence of uterine epithelial neoplasms in B6C3F1 mice (chloroethane 86%, bromoethane 56%) and a lower incidence for ethylene oxide (10%). The uterine neoplasms were classified as adenomas, adenocarcinomas, and squamous cell carcinomas for bromoethane, and as adenocarcinomas for both chloroethane and ethylene oxide. The adenocarcinomas and squamous cell carcinomas were invasive into the myometrium and the serosa, and metastasized to a wide variety of organs. Metastatic sites included most commonly the lung, lymph nodes, and ovary at unusually high rates of metastases (79% for chloroethane and 38% for bromoethane). Because of the dramatically high rates of uterine neoplasms (induced by chemicals given by the inhalation route) and metastases, a re-evaluation of the pathology and incidence data was undertaken. The earlier results were confirmed. The mechanism of uterine carcinogenesis by chloroethane, bromoethane and ethylene oxide is unclear.     

Urinary bladder neoplasms induced in BALB/c female mice with low doses of 2-acetylaminofluorene

Large numbers of female BALB/c mice were exposed to low levels of 2-acetylaminofluorene (2-AAF) in the diet. The dose response for urinary bladder neoplasms exhibited a shallow trend to low doses which increased sharply at the higher doses. The dose response over the entire dosage range gave the impression of a "threshold" type response below the mid-doses. Since the tumor incidence at the lower doses was less than 1.0 percent, relatively few urinary bladder tumors occurred. Thus, it was deemed advisable to re-examine noninvasive or early invasive bladder carcinomas and to study the dose response for only the "unequivocal" urinary bladder tumors. Examination of the dose response curves for "unequivocal" urinary bladder tumors showed a positive trend over the low end of the dose range. As a further check, an analysis was conducted on only invasive or metastasizing bladder carcinomas. In this case, there was no significant low dose trend. Thus, the shape of the low dose response curve for bladder carcinomas remains uncertain.     

Overview of the molecular biology of hepatocellular neoplasms and hepatoblastomas of the mouse liver

The molecular pathogenesis of chemically induced hepatocellular neoplasms and hepatoblastomas in the B6C3FI mouse is unclear but may involve alterations in the fi-catenin/Wnt signaling pathway as was recently described for human liver neoplasms. The objectives of this research were to characterize the mutation frequency and spectrum of P-catenin mutations and the intracellular localization of I-catenin protein accumulation in chemically induced hepatoblastomas and hepatocellular neoplasms. In the majority of the hepatoblastomas examined by immunohistochemical methods, both nuclear and cytoplasmic localization of P-catenin protein were detected, whereas in hepatocellular adenomas and carcinomas and normal liver only membrane staining was observed. Genomic DNA was isolated from paraffin sections of each liver tumor. P-catenin exon 2 (corresponds to exon 3 in humans) genetic alterations were identified in the majority of hepatoblastomas from exposed mice. Deletion mutations were identified more frequently than point mutations in hepatoblastomas. Hepatocellular adenomas and carcinomas from treated mice had mutations in exon 2 of the B-catenin gene which ranged from 32-43%, while 10% P-catenin mutations were detected in spontaneous neoplasms. By immunohistochemical methods cyclin Dl was observed in most nuclei of hepatoblastomas and strong expression of cyclin Dl was confirmed by Western analysis regardless of treatment. The cumulative data suggests that P-catenin mutations with upregulation of the B-catenin protein and Wnt signaling most likely increased cyclin Dl expression. Cyclin D1 may provide an advantage during tumor progression of hepatocellular neoplasms and hepatoblastomas. The review will also focus on other genes which are important in mouse and human liver tumors.     

Stereology, flow cytometry, and immunohistochemistry of follicular neoplasms of the thyroid gland

A retrospective analysis of surgically resected thyroid cold solitary nodules was performed by stereology, DNA flow cytometry, and immunohistochemistry in 15 follicular adenomas and 15 well-differentiated follicular carcinomas to determine if a differential diagnosis of both follicular neoplasms can be done exclusively from cytologic criteria. Between 150 and 200 tumor cell nuclei (TCN) were studied per case for their TCN profile area, perimeter, and density, as well as for stereologic estimates, including the new parameter, volume-weighted mean particle volume (Vv). Flow-cytometric analyses included measurement of the DNA index and the percentage of cells in S phase and G2M phase. The same tumors were examined for the expression of thyroglobulin and the intermediate filaments vimentin and keratin. Follicular adenomas and follicular carcinomas did not show any significant differences in stereologic estimates related to TCN size. Both groups included similar proportions of diploid and aneuploid neoplasms. Aneuploid follicular neoplasms showed a significantly greater area, perimeter, and volume of TCN as compared with diploid tumors, regardless of their histologic diagnosis. Follicular adenomas and follicular carcinomas expressed a similar staining pattern for the tested immunoreagents, with a few cases coexpressing vimentin and keratin. From our results, a differential diagnosis of follicular neoplasms cannot be performed on the basis of cytologic aspirates exclusively. Infiltration of capsula or vessels remains the only safe indicator of malignancy in the absence of metastases. The lack of cytologic differences suggests that some follicular adenomas are preinvasive carcinomas, not yet showing infiltrative growth at the time of resection.     

Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice.

The localization of surfactant apoprotein (SAP) and the Clara cell antigen(s) (CCA) was studied in naturally occurring and experimentally induced pulmonary hyperplasias and neoplasms by avidin-biotin peroxidase complex (ABC) immunocytochemistry. Lungs of B6C3F1 and A strain mice with naturally occurring lesions, B6C3F1 mice given injections of N-nitrosodiethylamine (DEN), BALB/c nu/nu or nu/+ mice exposed transplacentally on Day 16 of gestation to ethylnitrosourea (ENU), or BALB/c nu/+ mice exposed to ENU at 8-12 weeks of age were preserved in formalin or Bouin's fixative. After ABC immunocytochemistry, SAP was found in the cytoplasm of normal alveolar Type II cells; in the majority of cells in focal alveolar and solid hyperplasias originating in peribronchiolar or peripheral locations; and in solid, tubular, papillary, and mixed adenomas and carcinomas. The larger mixed-pattern neoplasms and small or large tubular neoplasms usually had the least number of cells with SAP. The majority of large papillary adenomas and carcinomas in BALB/c mice exposed to ENU and in untreated A strain mice contained SAP in the nuclei of many neoplastic cells but only in the cytoplasm of a few neoplastic cells. CCA was found in normal Clara cells of bronchi and bronchioles but not in any hyperplastic or neoplastic lesion of any mouse studied. This study provided immunocytochemical evidence that the vast majority of naturally occurring and experimentally induced pulmonary neoplasms of mice are alveolar Type II cell adenomas and carcinomas.     

Her-2 expression in cutaneous eccrine and apocrine neoplasms.

Cutaneous eccrine and apocrine glands have many histologic and immunologic similarities to ducts and acini of the breast. Thus, differentiating a primary cutaneous process from a metastatic breast carcinoma can be nearly impossible. In all, 10-34% of breast carcinomas overexpress HER-2 protein, a membrane-associated protein that functions in cell differentiation, adhesion and motility. As expression of this gene in cutaneous neoplasms has not been well characterized, we sought to determine HER-2 expression in a sample of benign and malignant cutaneous eccrine and apocrine neoplasms and to determine if there is value in using this protein expression in differentiating primary cutaneous from metastatic breast lesions. Totally, 85 primary cutaneous neoplasms and 11 cutaneous metastases from HER-2-positive breast carcinomas were retrieved from archived material at our institute. All cases were evaluated for HER-2 protein expression using the Dako Hercept Test kit. Membranous HER-2 staining was noted in three of the 85 cutaneous adnexal neoplasms: one hidrocystoma and two nodular hidradenomas. Seven of the 11 cutaneous metastases from HER-2-positive breast carcinomas maintained moderate-to-strong HER-2 expression. In conclusion, while 10-34% of breast carcinomas overexpress the HER-2 protein, only 3.5% of cutaneous apocrine and eccrine neoplasms in this study stained with the HER-2 antibody. These HER-2-positive cutaneous neoplasms typically do not pose a diagnostic dilemma in the setting of differentiation from breast metastasis. Additionally, although histologically these breast and cutaneous lesions may have morphologic similarities, the relative lack of HER-2 overexpression suggests that they are different nosologically. Finally, this study suggests that HER-2 protein expression can be a useful tool in differentiating a primary cutaneous appendageal neoplasm from HER-2 expressing metastatic breast carcinoma.     

Comparative characteristics of the stroma of renal adenomas and cancer

In a comparative study of 120 renal carcinomas and 10 adenomas it was found that in the adenomas the stroma as well as vessels were uniform with respect to distribution and degree of maturity and showed only scanty lymphoid infiltration, but that in the carcinomas, on the contrary, the distribution was irregular and lymphoid infiltration was much more abundant, particularly in areas of invasive growth. A close correlation was observed between stromal morphology and proliferative activity of the tumor. It is concluded that morphologic differences between these two kinds of tumor may be of use in diagnostic differentiation.     

Ultrastructure of hepatocellular neoplasms in aflatoxin B1 (AFB1)-initiated rainbow trout (Oncorhynchus mykiss).

The fine structure of hepatocellular neoplasms from aflatoxin B1 (AFB1)-initiated rainbow trout was studied by transmission electron microscopy. Large, usually uniform hepatic nuclei, large nucleoli, abundant, dilated rough-surfaced endoplasmic reticulum, and reduced glycogen storage were common findings in both hepatocellular adenomas and hepatocellular carcinomas. In addition, the presence of poorly developed microvilli in the space of Disse and in bile canaliculi, the occurrence of few or no bile preductule cells and a striking increase in the size and number of intercellular spaces characterized hepatocellular carcinomas. The three latter characteristics of hepatocellular carcinomas suggest loss of inter-relationships between hepatocytes and the microvascular system (sinusoids), between hepatocytes and the biliary system, and between individual hepatocytes, respectively. With respect to these parameters, adenomas were more similar to normal liver than to carcinomas.     

Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis

Nonpolypoid neoplasms, as well as ordinary polypoid tumours, are occasionally found in the colorectum. To clarify whether cell kinetic status affects the macroscopic morphology of colorectal neoplasms, we investigated proliferative indices (PI), apoptotic indices (AI), and the expression of apoptosis-related gene products. We examined 110 colorectal neoplasms comprised of 36 polypoid, 38 flat elevated and 36 depressed tumours. According to WHO’s criteria these tumours consisted of 61 adenomas with low grade dysplasia (LGD), 30 adenomas with high grade dysplasia (HGD) and 19 carcinomas with submucosal invasion. Apoptotic cells were detected by TUNEL staining. Proliferating cells and apoptosis-related gene products were assessed by immunohistochemistry for Ki-67, p53, Bcl-2, and Bax antigens. AI were closely associated with macroscopic morphology in adenomas but not in carcinomas. PI were relatively constant among the three macroscopic types in adenomas and carcinomas. Median AI values of polypoid, flat elevated and depressed tumours were 1.8%, 2.1% and 4.6% for adenomas with LGD, 0.8%, 2.4% and 6.2% for adenomas with HGD and 2.9%, 4.0% and 3.6% for carcinomas, respectively. Overall PI were significantly higher in carcinomas than in adenomas with LGD, whereas AI were not different. Although the incidence of expression was significantly higher in carcinomas for p53 and in adenomas for Bcl-2 than the others, the expression of apoptosis-related gene products (p53, Bcl-2 and Bax) was similar among polypoid, flat elevated and depressed tumours. Macroscopic morphology of colorectal adenomas is determined by the apoptosis not by proliferation, and high apoptosis found in depressed adenomas implies their low net growth. Received: 1 July 1999 / Accepted: 17 January 2000     

Immunocytochemical evaluation of neoplastic and non-neoplastic breast diseases with Mab A-80

Five hundred breast tissue samples from 404 cases were immunostained with A-80, a murine IgM Mab that recognizes a mucinous glycoprotein associated with exocrine differentiation. Samples included 196 primary breast carcinomas, 30 breast carcinoma metastases, 118 fibrocystic disease (FCD), and a further group of 84 samples of FCD from cases known to have breast carcinoma. These samples represented a broad spectrum of common and rare variants of carcinoma and FCD. Samples of fibroadenomas, lactating adenomas, cystosarcoma phylloides, gynecomastia, and normal breasts were similarly studied. The vast majority of carcinomas, 203/212 (95.7%) were immunoreactive; staining varied in extent and intensity, and was virtually unrelated to histologic type and to the presence or absence of recognizable glands. In samples including in-situ and infiltrating ductal or lobular carcinoma, reactivity was frequently stronger in the infiltrating components. No significant difference in reactivity between primary and metastatic carcinomas was noted. Of the group of 118 FCD, 27 were negative whereas 91 showed focal and weak staining. Seventy-two/84 FCD with associated carcinoma were immunostained; in 13 of those 72, staining was strong and extensive. Fibroadenomas, lactating adenomas, gynecomastia, and normal "resting" and lactating breast samples stained focally or not at all. Our findings indicate that Mab A-80 is an excellent immunohistochemical marker for the overwhelming majority of breast carcinomas whereas it marks weakly or not at all the majority of benign neoplasms and normal breast. Moreover, Mab A-80 recognizes a subset of FCD that includes proliferative variants associated with an increased incidence of carcinoma, and FCD in association with carcinoma. Questions regarding rare breast carcinomas that do not react with Mab A-80 remain unclear; yet, we believe that Mab A-80 is a highly promising marker of malignant and dysplastic breast epithelium.     

Anterior gradient-2 is overexpressed by fibrolamellar carcinomas

Anterior gradient-2 expression is critical in normal embryonic development. Aberrant expression of anterior gradient-2 in adult tissues has been linked to breast, prostate, esophageal, and pancreatic carcinoma. To define the role of anterior gradient-2 in primary hepatocellular neoplasms, we used tissue microarrays and examined protein expression in typical hepatocellular carcinomas (n = 44), fibrolamellar carcinomas (n = 12), and hepatic adenomas (n = 9). In nonneoplastic liver tissues, anterior gradient-2 was expressed in the septal-sized bile ducts and weakly in zone 3 hepatocytes in 11 (18%) of 61 cases. In tumors, anterior gradient-2 was overexpressed by only 1 (2%) of 44 hepatocellular carcinomas. In contrast, 6 (75%) of 8 fibrolamellar and 3 (75%) of 4 metastatic fibrolamellar carcinomas were positive. All 9 hepatic adenomas were negative. Further analysis of mRNA in fibrolamellar carcinomas identified 2 novel splice variants, but expression levels were very low. Sequencing of the anterior gradient-2 gene in fibrolamellar carcinomas identified several polymorphisms (refSNP Ids: rs6842, rs8071, rs1051905) but no mutations. In conclusion, anterior gradient-2 is overexpressed in the majority of fibrolamellar carcinomas but is only rarely overexpressed in hepatocellular carcinomas.     

Incidence and morphology of spontaneous thyroid tumours in different strains of rats

The incidence and morphology of thyroid neoplasms in different strains and stocks of rats are reported. The frequency of thyroid tumours varied to a great extent between different strains of rats. Low incidences were observed in DA/Han (0.85 per cent) and BDII/Han rats (1.25 per cent), while somewhat higher incidences occurred in Han:WIST rats (7.3 per cent). The highest number of thyroid tumour-bearing rats was found in the Han:SPRD stock (55.5 per cent). Thyroid neoplasms were classified histologically as polymorphofollicular adenomas, papillary adenomas, cystadenomas, polymorphofollicular carcinomas, papillary carcinomas, C-cell adenomas and C-cell carcinomas (medullary carcinomas). C-cell neoplasms were predominant in Han:SPRD rats (93.0 per cent), while the majority of tumour-bearing Han:WIST rats (61.4 per cent) had follicular tumours.     

Discerning malignancy in human adrenocortical neoplasms: utility of DNA flow cytometry and immunohistochemistry.

In order to evaluate more objective laboratory methods that may help practicing pathologists to discern malignancy in human adrenocortical neoplasms, we have examined cellular DNA content by flow cytometry and immunohistochemical distribution of c-myc, vimentin, proliferating cell nuclear antigen (PCNA), and epidermal growth factor receptor (EGFR) in 15 cases of human adrenocortical neoplasms (nine carcinomas and six adenomas). All of these examinations were performed on routinely processed surgical pathology specimens. All carcinoma cases met Weiss's histologic criteria. Seven of eight adrenocortical carcinomas demonstrated aneuploid DNA content, while all adenomas were diploid by flow cytometry. c-myc oncoprotein was observed both in cytoplasms and nuclei in all carcinomas but only in nuclei in adenomas. Vimentin was present in all carcinoma cases examined but was also observed in three of six cases of adenoma. There were no clinical or histologic differences between vimentin-positive and vimentin-negative adenomas. Immunoreactivity of PCNA and EGFR was observed in all the cases examined. There were no significant differences in distribution or patterns of immunoreactivity between adrenocortical carcinoma and adenoma. Therefore, we conclude that only DNA ploidy examined by flow cytometry and immunolocalization patterns of c-myc oncoprotein expression have any practical value in the pathologic evaluation of adrenocortical neoplasms. Careful morphologic and/or clinical studies are still considered to be the best available methods in discerning malignancy in resected human adrenocortical neoplasms.